Stable and controlled release formulations of neuregulin-4 analogs

Abstract

Enterically coated formulations of polypeptides are provided, which provides protection to the polypeptides in oral administration and preferably slow release of the polypeptides in vivo. The polypeptides are analogs of neuregulin-4 and exhibit one or more activities of neuregulin-4, including the ability to induce homodimerization of ErbB4 subunits, the specificity in inducing homodimerization of ErbB4 over heterodimerization of ErbB family subunits. A couple of polypeptides further possess stability against enzymatic degradation. The formulations can be used in subjects with an inflammatory condition in the gastrointestinal tract.

Description

STABLE AND CONTROLLED RELEASE FORMULATIONS OF NEUREGULIN-4 ANALOGS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 443,138, filed February 3, 2023, the entirety of which is hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under grant no. DK112435 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing submitted February 2, 2024, as an electronic file named “079065_000004WOPT_Sequence Listing”, having a size in bytes of 59,259 bytes and created on February 1, 2024, is hereby incorporated by reference. FIELD OF INVENTION

[0004] This invention relates to polypeptide compositions provided in an acid-stable and controlled release formulations. BACKGROUND

[0005] Neuregulins (NRGs) comprise a family of epidermal growth factor (EGF)-like signaling molecules involved in cell-cell communication during development and disease. The neuregulin family of ligands has four members: NRG-1, NRG-2, NRG-3, and NRG-4. Neuregulins transmit their signals to target cells by interacting with transmembrane tyrosine kinase receptors of the ErbB family. The ERBB family of receptor tyrosine kinases, namely the EGF receptor (EGFR), ERBB2 / HER2 / Neu, ERBB3 / HER3, and ERBB4 / HER4, regulate cellular proliferation, differentiation, cell survival and programmed cell death depending on the tissue context in a growth factor-dependent manner.

[0006] It is an objective of the present invention to provide formulations of peptidic analogs or variants of NRG-4 for protection against acid degradation and delivery to the gut.

[0007] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. SUMMARY OF THE INVENTION 4878-9453-5582.4 Page 1 of 80079065-000004WOPT

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0009] Various embodiments provide enteric coated compositions, wherein enteric coating imparts protection to core of the composition in a low pH environment of 3 or less while capable of releasing the core at a pH of 5.5 or higher, and wherein the core contains one or more polypeptides being neuregulin 4 (human) analogs, derivatives, or variants.

[0010] In some embodiments, an enteric coated composition comprises a core in the form of a particle and an enteric coating for the core, wherein the particle comprises a polypeptide and microcrystalline cellulose, and wherein the enteric coating imparts protection to the core so that the core is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher, said polypeptide comprising an amino acid having a structure of Formula I or a variant of Formula I having at least 90% or 95% sequence identity thereto.

[0011] In some embodiments, an enteric coated composition is in the form of a tablet and comprises an enteric coating on a compressed formulation, wherein the compressed formulation comprises a polypeptide, microcrystalline cellulose, and optionally a binding agent, and wherein the enteric coating imparts protection to the core so that the core is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher, said polypeptide comprising an amino acid having a structure of Formula I or a variant of Formula I having at least 90% or 95% sequence identity thereto, and said enteric coating comprising a copolymer derived from methacrylic acid and ethyl acrylate.

[0012] In some embodiments, the particle further comprises a binding agent selected from the group consisting of hydroxypropyl cellulose, calcium chloride, polyvinyl alcohol, hypromellose, magnesium stearate, and a combination thereof.

[0013] In some embodiments, the enteric coating comprises a copolymer derived from methacrylic acid and ethyl acrylate.

[0014] In some embodiments, the polypeptide is at least 0.05%, or 0.1-0.2%, 0.2%-0.3%, 0.4%-0.5%, or more by weight of the enteric coated composition.

[0015] In some embodiments, the core is in the form of a microparticle or a nanoparticle.

[0016] Dosage forms of a pharmaceutical composition are also provided, which comprise a shell and a plurality of one or more of the enteric coated compositions. In some embodiments, the dosage form is a capsule, and the shell of the capsule comprises gelatin.

[0017] In some embodiments, the polypeptide disclosed herein particularly for the enteric coated compositions or dosage forms, that is the polypeptide having a structure of Formula I or being a variant of Formula I, is not any of SEQ ID NO:1 (human NRG-4(5-50)), SEQ ID NO:30 (human4878-9453-5582.4Page 2 of 80079065-000004WOPTNRG4(1-62)), EGF, human NRG-3, rat NRG-4, mouse NRG-4, gorilla NRG-4, and chimpanzee NRG- 4.

[0018] In some embodiments, a variant of Formula I has at least one biochemical activity exhibited by a polypeptide selected from the group consisting of Formula I and SEQ ID NO:1. The biochemical activities include but are not limited to any one of more of inducing homodimerization of ErbB4 subunits, having specificity in inducing homodimerization of ErbB4 subunits over homodimerization of EGFR subunits by a difference in EC50 of at least 1,000 fold, having at least 90% stability, or more stable, compared to neuregulin-4, with respect to resisting enzymatic degradation in intestinal fluid or a simulated intestinal fluid, having at least 90% stability, or more stable, compared to neuregulin-4, with respect to resisting enzymatic degradation in gastric fluid or a simulated gastric fluid.

[0019] Methods for preparing the enteric coated compositions are also provided. In some embodiments, a preparation method includes mixing the polypeptide, the microcrystalline cellulose, and a binding agent in a slurry; generating particles from the slurry by granulation, extrusion, and / or spheronization; and spraying coating a pH-sensitive polymer on the particles. In some embodiments, a preparation method includes mixing the polypeptide, the microcrystalline cellulose, and the binding agent in a slurry; compressing the slurry in a form of a tablet, thereby forming a compressed formulation; and spraying a pH-sensitive polymer on the tablet, or dipping the tablet in a solution or suspension of the pH-sensitive polymer. Preferably, the pH-sensitive polymer imparts protection to the particle or tablet so that the particle or tablet is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher.

[0020] Methods are also provided for using the enteric coated compositions for treating, reducing severity, or inhibiting diseases in a mammalian subject, especially human. Exemplary diseases or conditions to be treated include an inflammatory condition in gastrointestinal tract (such as ulcerative colitis, Crohn’s disease, necrotizing enterocolitis, or radiation-induced gastrointestinal injury), acute lung injury, a metabolic disorder (such as diabetes or non-alcoholic steatohepatitis).

[0021] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. BRIEF DESCRIPTION OF THE FIGURES

[0022] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0023] Figure 1 shows neuregulin-4 (NRG-4 (1-62) amino acid sequence (SEQ ID NO:30) with disulfide linkages indicated.

[0024] Figure 2 illustrates the principle of the PATHHUNTER® Kinase Dimerization assays (Prior Art). The PATHHUNTER® Dimerization assay detects ligand induced dimerization of two subunits of a receptor-dimer pair. The cells have been engineered to co-express one receptor subunit4878-9453-5582.4Page 3 of 80079065-000004WOPTfused to Enzyme Donor (ED), and a second dimer partner fused to Enzyme Acceptor (EA). Cytoplasmic tail may have been deleted from one or both receptors. Binding of an agonist to one receptor subunit induces it to interact with its dimer partner, forcing complementation of the two enzyme fragments. This results in the formation of a functional enzyme that hydrolyzes a substrate to generate a chemiluminescent signal. The approach is applicable to assess the function of biologics and / or antibody therapeutics with respect to receptor kinase homodimers or heterodimers.

[0025] Figures 3 and 4 depict the result of metabolism study of NRG-4, NRG-4 analogs, and two controls, desmopressin and somatostatin, in the presence of simulated intestinal fluid (SIF) containing chymotrypsin. (FIG.3) Analogs (SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:15) that have comparable stability to NRG-4, and analogs (SEQ ID NO:17, SEQ ID NO:18) that are less stable than NRG-4. (FIG. 4) Analogs (SEQ ID NO:8, SEQ ID NO:1, SEQ ID NO:16, SEQ ID NO:22) that are more stable than NRG-4. N= 2 replicates.

[0026] Figure 5 depicts the result of metabolism study of NRG-4, NRG-4 analogs, and two controls in the presence of simulated intestinal fluid (SIF) containing trypsin.

[0027] Figures 6A and 6B depict the results of metabolism study of NRG-4 and NRG-4 analogs (6A), and of two controls (6B), in the presence of simulated gastric fluid (SGF) containing pepsin.

[0028] Figure 7A-7B depict the stability of NRG-4 analogs in mouse plasma (7A), and stability of NRG-4 analogs in human plasma (7B).

[0029] Figure 8 depicts a comparison of Peptide Purity of selected NRG-4 analogs after 2 weeks of storage at 2-8 °C and at 20 °C.

[0030] Figure 9 depicts an NRG-4 Efficacy Study in a DSS-induced colitis mouse model using intra-cecal administration of the test article and shows the body weight change.

[0031] Figure 10 depicts the Colitis Severity and Stool Consistency Scores (Day 12) for the NRG-4 Study. Animals underwent video endoscopy on Day 12 to assess colon inflammation and were assigned colitis severity and stool consistency scores. Statistical significance was determined by Kruskal-Wallis test with Dunn’s multiple comparisons test used to compare all groups to Group 2. ****p<0.0001. Data is presented as mean ± SEM. n=8-15 per group.

[0032] Figure 11 depicts the Body Weight Change for NRG-4 Analog 1 (SEQ ID NO:22) Efficacy Study in a DSS-induced colitis mouse model using intra-cecal administration of the test article. Animals were weighed daily, and body weight change as compared to Day 0 (Figure 11, upper) and Day 5 (Figure 11, lower) were calculated. The AUC was calculated using the trapezoidal rule transformation and is shown in the inset. Statistical significance between groups was determined by one-way ANOVA with Dunnett’s multiple comparisons test used to compare all groups to the vehicle- control group. **p<0.01. Data is presented as mean ± SEM. n=8-15 per group.4878-9453-5582.4Page 4 of 80079065-000004WOPT

[0033] Figure 12 depicts the composite DAI Scores for the NRG-4 Analog 1 Study. Animals were monitored for body weight loss, diarrhea, and blood in stool; each of these parameters was assigned a score and these scores were used to generate a daily composite DAI score. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison post-test used to compare all groups to Group 2. Data is presented as mean ± SEM. n=8-15 per group.

[0034] Figure 13 depicts the Colitis Severity and Stool Consistency Scores (Day 12) Animals underwent video endoscopy on Day 12 to assess colon inflammation and were assigned colitis severity and stool consistency scores. Statistical significance was determined by Kruskal-Wallis test with Dunn’s multiple comparisons test used to compare all groups to Group 2. **p<0.01. Data is presented as mean ± SEM. n=8-15 per group.

[0035] Figure 14 depicts the Colitis Severity and Stool Consistency Scores (Day 19). Animals underwent video endoscopy on Day 19 to assess colon inflammation and were assigned colitis severity and stool consistency scores. Statistical significance was determined by Kruskal-Wallis test with Dunn’s multiple comparisons test used to compare all groups to Group 2. Data is presented as mean ± SEM. n=8-15 per group.

[0036] Figure 15A depicts the Colon Measurements at Euthanasia. Animals were euthanized on Day 19 and the colon was measured and weighed to enable calculation of the weight:length ratio. Data is presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparison post-test used to compare all groups to Group 2. n=8-15 per group. Figure 15B depicts that SEQ ID NO:22 significantly improved key end points in mouse DSS studies in terms of mean histopathology scores.

[0037] Figure 16A depicts an exemplary manufacturing process of enteric-coated NRG-4- microcrystalline cellulose (MCC) beads. Figure 16B depicts a schematic showing an oral drug product of capsules filled with enteric coated, polypeptide-MCC beads for colonic and intestinal delivery of the polypeptide. Alternatively for high drug doses, an enterically coated tablet form using the same MCC and binder may be orally ingested.

[0038] Figure 17 depicts Kaplan Meier Curves (A) Parent NRG-4 vs SEQ ID NO:22 dose groups and (B) Parent NRG-4 vs Grouped SEQ ID NO:22 Treatment in T cell-mediated chronic colitis model.

[0039] Figure 18 depicts the percent weight loss for NRG-4 (1500 μg / mg) and SEQ ID NO:22 (600 μg / kg) vs Vehicle. Lower: AUC for the different cohorts.

[0040] Figure 19 depicts disease activity index for different treatment groups. Lower: AUC for the curves starting from Day 0.

[0041] Figure 20 depicts Video Endoscopy Scores (Day 21). Data is presented for individual animals, as well as mean ± SEM. n=13-15 per group.

[0042] Figure 21 depicts Stool Consistency Score (Day 42).4878-9453-5582.4Page 5 of 80079065-000004WOPT

[0043] Figure 22A depicts Mouse Proximal Colon Component Histopathology Scores. TA = SEQ ID NO:22.

[0044] Figure 22B depicts Mouse Distal Colon Component Histopathology Scores. TA = SEQ ID NO:22.

[0045] Figure 23 depicts stability testing of enteric coated, SEQ ID NO:22-MCC beads at 25°C / 60% relative humidity (RH), and 40°C / 75% RH.

[0046] Figure 24 depicts a diagram in purification of the polypeptide whose amino acid sequence is shown in SEQ ID NO:22. DESCRIPTION OF THE INVENTION

[0047] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0048] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0049] “Enteric coating” is a polymer material or materials which encases the medicament core. The polymeric enteric coating material does not contain an active compound, i.e. an therapeutically active polypeptide disclosed herein. Preferably, a substantial amount or all of the enteric polymer coating material is dissolved before the medicament or therapeutically active polypeptide is released from the dosage form, so as to achieve delayed dissolution of the medicament core. A suitable pH-sensitive polymer is one which remain intact in the lower pH environs of the stomach but will dissolve with intestinal juices at the higher pH levels (pH greater than 5.5 or higher), such as within the small intestine and therefore permit release of the pharmacologically active substance in the regions of the small intestine and not in the upper portion of the GI tract, such as the stomach.

[0050] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.4878-9453-5582.4Page 6 of 80079065-000004WOPT

[0051] “N-terminus” refers to the first amino acid residue in a protein or polypeptide sequence. Generally, the N-terminal residue contains a free Į-amino group, unless covalently linked to a non- amino acid moiety (e.g., lipid moiety, optionally with a non-amino acid linker) as disclosed herein.

[0052] “C-terminus” refers to the last amino acid residue in a protein or polypeptide sequence. Generally, the C-terminal residue contains a free carboxylate group, unless covalently linked to a non- amino acid moiety (e.g., lipid moiety, optionally with a non-amino acid linker) as disclosed herein.

[0053] “Polymer” refers to a macromolecule having repeating units connected by covalent bonds. Polymers can be hydrophilic, hydrophobic or amphiphilic. Hydrophilic polymers are substantially miscible with water and include, but are not limited to, polyethylene glycol. Hydrophobic polymers are substantially immiscible with water and include, but are not limited to, polybutadiene and polystyrene. Polymers include homopolymers, random copolymers, and block copolymers.

[0054] “Hydrophobic moiety” refers to polymers or small molecules that are hydrophobic. Examples of hydrophobic moieties include, but are not limited to, hydrophobic polymers such as lipid moieties, as well as polybutadiene and polystyrene.

[0055] “Lipid moiety” refers to a moiety having at least one lipid. Lipids are small molecules having hydrophobic or amphiphilic properties and are useful for preparation of vesicles, micelles and liposomes. Lipids include, but are not limited to, fats, waxes, fatty acids, cholesterol, phospholipids, monoglycerides, diglycerides and triglycerides. The fatty acids can be saturated, mono-unsaturated or poly-unsaturated. Examples of fatty acids include, but are not limited to, butyric acid (C4), caproic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), isostearic acid (C18), oleic acid (C18), vaccenic acid (C18), linoleic acid (C18), alpha-linoleic acid (C18), gamma-linolenic acid (C18), arachidic acid (C20), gadoleic acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20), behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22), lignoceric acid (C24) and hexacosanoic acid (C26). The lipid moiety can include several fatty acid groups using branching groups such as lysine and other branched amines. In further embodiments, the hydrophobic moiety has a structure of HO2CíRíC(=O)í, where R can be aliphatic or aromatic, saturated or unsaturated, linear or branched molecule; which is derived from dicarboxylic acids (di-acid). Exemplary dicarboxylic acids include, but are not limited to, malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (or tridecanedioic acid, C13), tetradecanedioic acid (C14), pentadecanedioic acid (C15), thapsic acid (or hexadecanedioic acid, C16), heneicosadioic acid (C21), docosanedioic acid (C22), and triacontanedioic acid (C30).

[0056] “Alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl groups can have up to 24 carbons atoms and include heptyl, octyl, nonyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl,4878-9453-5582.4Page 7 of 80079065-000004WOPTicosyl, and the like. Alkyl can include any number of carbons such as C6 to C20, C6 to C18, C6 to C16, C8 to C24, C8 to C22 and C8 to C20. Alkyl groups can be substituted with substituents including fluorine groups.

[0057] ĀAcylā refers to a carbonyl radical (i.e., CőO) substituted with an alkyl group as defined above. The number of carbon atoms indicated for an acyl group includes the carbonyl carbon and the alkyl carbons. Acyl groups can have up to 24 carbons atoms and include heptanoyl (heptoyl), octanoyl (octoyl), nonanoyl (nonoyl), decanoyl (decoyl), dodecanoyl (dodecoyl), tridecanoyl (tridecoyl), tetradecanoyl (tetradecoyl), pentadecanoyl (pentadecoyl), hexadecanoyl (hexadecoyl), heptadecanoyl (heptadecoyl), octadecanoyl (octadecoyl), nonadecanoyl (nonadecoyl), icosanoyl (eicosanoyl or icosoyl), and the like. Acyl groups can be substituted with substituents including fluorine groups. Acyl can include any number of carbons such as C6 to C20, C6 to C18, C6 to C16, C8 to C24, C8 to C22 and C8 to C20.

[0058] “Amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, Ȗ-carboxyglutamate, and 0-phosphoserine. Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0059] Single letter and three letter abbreviations for naturally occurring amino acids described in the invention are defined below in the following Table.4878-9453-5582.4Page 8 of 80079065-000004WOPT

[0060] “Conservatively modified variants” apply to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical amino acid sequence or an amino acid sequence with at least 90% identity to the original amino acid sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0061] The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D-enantiomeric and L-enantiomeric amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics.

[0062] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid (i.e., hydrophobic, hydrophilic, positively charged, neutral, negatively charged). Exemplified hydrophobic amino acids include valine, leucine, isoleucine, methionine, phenylalanine, and tryptophan. Exemplified aromatic amino acids include phenylalanine, tyrosine and tryptophan. Exemplified aliphatic amino acids include serine and threonine. Exemplified basic amino acids include lysine, arginine and histidine. Exemplified amino acids with carboxylate side-chains include aspartate and glutamate. Exemplified amino acids with carboxamide side chains include asparagines and glutamine.4878-9453-5582.4Page 9 of 80079065-000004WOPT

[0063] The following eight groups each contain amino acids that are conservative substitutions for one another (see, e.g., Creighton, Proteins (1984)): 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0064] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). Unless otherwise indicated, three-letter and single-letter abbreviations of amino acids refer to the L-isomeric form of the amino acid in question. The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dasp, (d)Asp or D-Asp; Dphe, (d)Phe or D-Phe). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the peptide. D- amino acids may be indicated as customary in lower case when referred to using single-letter abbreviations.

[0065] The 20 “standard,” natural amino acids are listed above and include Proline. The “non- standard,” natural amino acids are pyrrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many noneukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria and chloroplasts). “Unnatural”, “non- canonical” or “non-natural” amino acids are non-proteinogenic amino acids (i.e., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include ȕ-amino acids (ȕ3and ȕ2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine, tryptophan and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha- methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. According to certain embodiments, a peptide comprises an intramolecular bond between two amino acid residues present in the peptide. It is understood that the amino acid residues that form the bond will be altered somewhat when bonded to each other as compared to when not bonded to each4878-9453-5582.4Page 10 of 80079065-000004WOPTother. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state.

[0066] In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib (Į- aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), Ȗ-Glu (Ȗ- glutamic acid), Gaba (Ȗ-aminobutanoic acid), ȕ-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8- amino-3,6-dioxaoctanoic acid), Abu (2-amino butyric acid), ȕhPro (ȕ-homoproline), ȕhPhe (ȕ- homophenylalanine) and Bip (ȕ,ȕ diphenylalanine), and Ida (Iminodiacetic acid).

[0067] One of skill in the art will appreciate that certain amino acids and other chemical moieties are modified when bound to another molecule. For example, an amino acid side chain may be modified when it forms an intramolecular bridge with another amino acid side chain, e.g., one or more hydrogen may be removed or replaced by the bond. Accordingly, as used herein, reference to an amino acid or modified amino acid present in a Peptide of the Invention is meant to include the form of such amino acid or modified amino acid present in the peptide both before and after forming the intramolecular bond.

[0068] The term “NH2,” as used herein, can refer to a free amino group present at the amino terminus of a polypeptide. The term “OH,” as used herein, can refer to a free carboxy group present at the carboxy terminus of a peptide. Further, the term “Ac,” as used herein, refers to Acetyl protection through acylation of the C- or N-terminus of a polypeptide. In certain peptides shown herein, the NH2locates at the C-terminus of the peptide indicates an amino group.

[0069] The term “carboxy,” as used herein, refers to –CO2H.

[0070] The term “isostere replacement,” as used herein, refers to any amino acid or other analog moiety having chemical and / or structural properties similar to a specified amino acid. In certain embodiments, an isostere replacement is a conservative substitution or an analog of a specified amino acid.

[0071] The term “cyclized,” as used herein, refers to one part of a peptide molecule being linked to another part of the peptide molecule to form a closed ring, such as by forming a disulfide bridge or thioether bond.

[0072] The terms “component” or “subunit,” are used interchangeably and refer to one of a pair of peptide monomers that are joined to form a Peptide of the Invention.

[0073] The term “linker moiety,” as used herein, refers broadly to a chemical structure that is capable of linking or joining together two peptide subunits or linking a peptide to another group, for example, a fatty acid.

[0074] Human NRG-4 is related to the EGF family of peptides. Unlike many of its family members (e.g. NRG-1, HB-EGF, NRG-2), NRG-4 is a highly specific ligand for the ErbB4 receptor. It4878-9453-5582.4Page 11 of 80079065-000004WOPTis a 62 amino acid peptide with three disulfide linkages. The bioactivity of the molecule is derived from its EGF-like domain that contains the conserved cysteines which are linked in the specific manner shown in Figure 1. NRG-4 activates the ErbB4 receptor in cell-based assays and is a partial agonist compared to the mixed ligand NRG-1. We have used simulated gastric fluid (SGF) and intestinal fluid studies to identify GI peptidase-sensitive sites in parent NRG-4 sequence.

[0075] While not wishing to be bound by any particular theory we believe that the lipidated analogs can bind fatty acid transporters in enterocytes, leading to greater residence time in the gastrointestinal (GI) tract, and in some embodiments, are used to prepare an injectable formulation, which would have an extended duration of action via fatty acid binding to albumin. Enteric Coated Particles

[0076] Compositions are provided, comprising a core in the form of a particle (or bead, beadlet, granule, or pellet) and an enteric coating for the core. In various embodiments, the enteric coating imparts protection to the core so that the core is afforded protection in a low pH (acidic) environment while capable of releasing medicament at a higher pH (e.g., neutral or basic) environment, said medicament comprising a polypeptide disclosed herein. In various embodiments, a polypeptide disclosed herein is mixed or embedded or in combination with MCC and optionally a binding agent or another excipient, to form the core of the composition, and the core is in the form of a particle. In some embodiments, a quantity of one or more polypeptides disclosed herein is bound to the surface of a MCC particle (or bead or beadlet) via hydroxypropyl cellulose as a binder. In other embodiments, the core is in the form of a tablet, comprising a quantity of particles (or beads, beadlets, granules, or pellets). In further embodiments, the core in the form of a tablet is in a size that fits into a human mouth for oral ingestion.

[0077] In some aspects, the particle can have a diameter of from about 0.1 to about 5 mm, preferably from about 0.1 to about 1 mm, and preferably from about 0.2 to about 0.5 mm. In some aspects, the particle is about 300 μm. In some aspects, the particle is a microparticle.

[0078] In some embodiments, MCC is about 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-97%, or 97%-99%, or up to 99.9% (by weight) of the core / particle, and the polypeptide is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or 0.5%-1%, about 2%, about 3%, about 4%, about 5%, 5%-10%, 10%-20%, 20%-30% (by weight) of the core / particle.

[0079] A further binding agent (or binder) may be present in the core or mixed with the core, e.g., in an amount ranging from about 0 to about 10% by weight of the enteric coated composition. Exemplary binding agents are calcium chloride, polyvinyl alcohol, hypromellose or hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose, magnesium stearate, sodium carboxymethylcellulose, starch, polyvinylpyrrolidone, potassium alginate, and sodium alginate. In some embodiments, the core includes one or a quantity of MCC particles (or beads, beadlets, granules, or pellets), and the binding agent (or binder) includes hydroxypropyl cellulose, for binding of a4878-9453-5582.4Page 12 of 80079065-000004WOPTpolypeptide disclosed herein. In some embodiments, the core consists of one or a quantity of MCC particles (or beads, beadlets, granules, or pellets), and the binding agent (or binder) consists of hydroxypropyl cellulose, for binding of a polypeptide disclosed herein.

[0080] Enteric coatings are used to arrest the release of medicament (of the core) from orally ingestible dosage forms. In various embodiments, the enteric coatings comprise a pH-sensitive (or acid- stable in preferable embodiments) polymer, which, as a coating, imparts protection to the core so that the core is afforded protection in a low pH environment of 3 or less (e.g., about pH 2, or gastric fluid, or a simulated gastric fluid such as a solution of sodium chloride at a pH of about 1.2) while capable of releasing medicament at a pH of 5.5 or higher (e.g., about pH 5.5, pH 6-7, or intestinal fluid or gut environment).

[0081] Exemplary pH-sensitive polymers for enteric coating are copolymers of methacrylic acid and methacrylic acid methylester (EUDRAGITs), neutral copolymer of polymethacrylic acid esters (EUDRAGIT L30D), hydroxypropyl methylcellulose phthalate HP50 (HPMCP-HP50), HP55, HP55S, polyvinyl acetate phthalate, cellulose acetate phthalate, or further disclosed in U.S. Pat. Nos.5,225,202 and 5,536,507, which are incorporated by reference fully herein. The EUDRAGIT-type polymers include, for example, EUDRAGIT FS 30D, L 30 D-55, L 100-55, L 100, L 12.5, L 12.5 P (also referred to as L 12,5 P), RL 30 D, RL PO, RL 100, RL 12.5, RS 30 D, RS PO, RS 100, RS 12.5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12.5, and S 12.5 P. Similar polymers include KOLLICOAT MAE 30 DP and KOLLICOAT MAE 100 P. In some embodiments, one or more of EUDRAGIT FS 30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL PO, RL 100, RL 12,5, RS 30 D, RS PO, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12,5 S 12,5 P, KOLLICOAT MAE 30 DP and KOLLICOAT MAE 100 P is used. Eudragit is a synonym known for polymethacrylates; as well as various other synonyms for polymethacrylates such as Acryl-EZE, Acryl-EZE MP, Eastacryl 30D; Kollicoat MAE 30 D; Kollicoat MAE 30 DP; and polymeric methacrylates. Polymethacrylates are synthetic cationic and anionic polymers of dimethylaminoethyl methacrylates, methacrylic acid, and methacrylic acid esters in varying ratios. Detailed review of Eudragit is described by Patra et al., Future Journal of Pharmaceutical Sciences, Volume 3, Issue 1, June 2017, Pages 33-45.

[0082] In some embodiments, an enteric coating solution of EUDRAGIT L-30-D 55 is used in forming the enteric coated composition. EUDRAGIT L-30-D 55 is an aqueous acrylic resin dispersion, an anionic copolymer derived from methacrylic acid and ethyl acrylate with a ratio of the free carboxyl groups to the ester groups of approximately 1:1, and a mean molecular weight of approximately 250,000 g / mol (or based on SEC method a weight average molar mass of approximately 320,000 g / mol), usually supplied as an aqueous dispersion containing 30% w / w of dry lacquer substance.

[0083] In other embodiments, the enteric coating comprises a polymer having dissociable carboxylic groups, such as derivatives of cellulose, including hydroxypropylmethyl cellulose phthalate,4878-9453-5582.4Page 13 of 80079065-000004WOPTcellulose acetate phthalate and cellulose acetate trimellitate and similar derivatives of cellulose and other carbohydrate polymers.

[0084] In some embodiments, the enteric polymer coating is in an amount to be substantially dissolved during the transit time within the small intestine. Generally, the pH of the small intestine gradually increases from about 4.5 to about 6.5 in the duodenal bulb to about 7.2 in the distal portions of the small intestine (ileum); and a small intestine transit time is about 3 hours.

[0085] In some embodiments, the enteric coating is present in a weight fraction within the range of from about 5% to about 10%, about 10% to about 30%, about 30% to about 50%, or about 50% to 60% of the enteric coated composition. For example, the enteric coating in an amount of about 5% to about 30% may permit release of the core in the small intestine, and increasing the amount to approximately 60% may permit release in the colon.

[0086] Other excipients may also be present in the enteric coated composition. For example, a plasticizer may be present in the enteric coating, such as diethyl phthalate, triethyl citrate, triacetin, tributyl sebecate, or polyethylene glycol. The core may also include a disintegrant or a swelling agent. The enteric coated composition may also include an anti-adherent (anti-agglomerant), such as talc, magnesium stearate, or fumed silica.

[0087] In some embodiments, the enteric coated composition further includes, or is accompanied by, a protease inhibitor, such as aprotinin, an absorption enhancer, or a mucosa adhesive polymer. Dosage form

[0088] In further embodiments, enteric-coated particles are prepared for oral delivery of the polypeptide(s) in capsule dosage form. Upon oral ingestion the capsule shell dissolves allowing the contents in the capsule to be exposed to the gastric contents.

[0089] In some embodiments, a dosage form is a capsule, wherein dry powder or pellets of a quantity of the enteric coated particles disclosed herein are contained within a shell. In various aspects, the shell is made from hard gelatin or hydroxypropyl methylcellulose (or hypromellose for short). In various aspects, the capsule is a hard gelatin capsule; or the shell is made from hard gelatin. In various aspects, a hard gelatin capsule comprises gelatin and water, and optionally colouring and opacifying agents. In various aspects, the hard gelatin capsule for liquid filling is identical in composition to the capsule used for filling powders.

[0090] In some embodiments, a dosage form is a soft gelatin capsule, wherein semisolid or liquid comprising the enteric coated particles disclosed herein are contained within a shell made from a relatively more flexible, plasticized gelatin film. In various aspects, a soft gelatin capsule contains a plasticizer in addition to gelatin and water. For example, glycerol can be used as a plasticizer at a level of approx. 30% or another level of choice. Usually, the moisture uptake of soft gelatin capsules plasticized with a plasticizer such as glycerol is considerably higher than that for hard gelatin capsules;4878-9453-5582.4Page 14 of 80079065-000004WOPTand the permeability of the gelatin shell without plasticizer is lower than that of the soft gelatin capsule with plasticizer.

[0091] Preferably with the capsules, the fill material does not penetrate into the zone between the body and cap before the sealing operation, so as to ensure an efficient sealing process. In some embodiments, capsules are sealed via banding using a gelatin band. In some embodiments, capsules are sealed using a hydroalcoholic solution, such as a 50:50 water / ethanol mixture. For example, a hard gelatin capsule sealing process includes: spraying a hydroalcoholic solution onto join between capsule halves, wherein capillary action draws liquid into the space between body and cap; removing excess fluid by suction; applying gentle heat (e.g., approx. 45°C) to complete the melting (e.g., over a period of about one minute), so that the two gelatin layers are fused together to form a complete 360° seal; and allowing the capsules to return to room temperature, i.e., gelatin setting or hardening process.

[0092] Further gelatin capsules in dosage forms, such as size “1” or “0” hard gelatin capsules, are available in the database of the U.S. Food & Drug Administration. Capsules suitable for filling and administration in rodents, large animals and humans are available with vendors, e.g., www.torpac.com, and for drug products for human health. In some aspects, size M capsules are suitable for mice; and the larger size 9 capsules are used for rats. For human use, capsules are manufactured in different sizes to accommodate different volumes or amounts of dry drug material. Eight different hard gelatin capsules, identified by sizes #000, #00, #0, #1, #2, #3, #4 & #5 are used in the pharmaceutical industry, wherein the 000 size has the largest dimensions and the #5 capsule has the smallest size. The size of the capsule that is chosen for human use will depend on factors such as required therapeutic dose and MCC bead drug load.

[0093] In various embodiments, in the hard gelatin capsule process, the capsule is pre- fabricated and supplied empty, whereas in the soft gelatin capsule process the encapsulation and filling take place simultaneously.

[0094] In some aspects, the shell comprises gelatin, and a plurality of enteric coated MCC- based particles are contained within the shell, wherein each enteric coated MCC-based particle comprises: a MCC particle, a quantity of a polypeptide, a binder, and an enteric coating at the outermost layer. In further aspects, the shell comprises gelatin, and a plurality of enteric coated MCC-based particles are contained within the shell, wherein each enteric coated MCC-based particle comprises: one MCC microparticle with a diameter of about 200 μm – 500 μm, a quantity of a polypeptide bound on the surface of the MCC microparticle via a binder including hydroxypropyl cellulose, and an enteric coating layer comprising a copolymer derived from methacrylic acid and ethyl acrylate (with a ratio of the free carboxyl groups to the ester groups being approximately 1:1) as the outermost layer of the enteric coated MCC-based particle.

[0095] In some aspects, gelatin capsules filled with enteric-coated, NRG-4 peptidic analog- MCC beads can be used for oral dosing for animal studies and for human use to treat disease. The4878-9453-5582.4Page 15 of 80079065-000004WOPTcapsules allow for accurate dosing of any type of dry material (powder, granule, small tablets etc.) and a variety of liquids and pastes. Gelatin itself is physiologically inert. Gelatin capsules dissolve rapidly and completely in the stomach to release the enterically coated drug loaded MCC beads. The beads then transit the stomach to the intestinal tract, the enteric coat dissolves at pH 5.5 and above to initiate the process of peptide dissolution and drug release.

[0096] Other embodiments provide that a dosage form is a tablet, which comprises an outer coating layer coated on a compressed formulation, wherein the outer coating layer comprises an enteric coating polymer derived from methacrylic acid and ethyl acrylate (with a ratio of the free carboxyl groups to the ester groups being approximately 1:1) (EUDRAGIT L30D-55), and the compressed formulation comprises a polypeptide disclosed herein mixed or embedded or in combination with MCC and optionally a binding agent such as hydroxypropyl cellulose. In some aspects, the dosage form as a tablet does not contain a gelatin-based shell.

[0097] In certain embodiments, the dosage form further comprises or is accompanied by a carrier, a diluent or an excipient. A pharmaceutically acceptable carrier, diluent or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

[0098] In certain embodiments, the dosage form further comprises or is accompanied by absorption enhancers that are also widely associated with other carriers to improve oral bioavailability by transporting of peptides and proteins across the intestinal mucosa. Such substances can be added as excipients or incorporated to form non-specific interactions with the intended polypeptide. Dietary components and / or other naturally occurring substances affirmed as enhancing tight junction permeation and as Generally Recognized As Safe (GRAS) include, e.g., acylglycerides, acylcarnitines, bile salts, and medium chain fatty acids. Sodium salts of medium chain fatty acids (MCFAS) function as permeation enhancers. The most extensively studied MCFAS is sodium caprate, a salt of capric acid, which comprises 2-3% of the fatty acids in the milk fat fraction.

[0099] In certain embodiments of the dosage form, a permeation enhancer is combined with polypeptide wherein the permeation enhancer comprises at least one of a medium chain fatty acid, a long-chain fatty acid, a bile salt, an amphiphilic surfactant, and a chelating agent. In certain embodiments, medium-chain fatty acid salts promote absorption by increasing paracellular permeability of the intestinal epithelium. In certain embodiments, a permeation enhancer comprising sodium N [hydroxybenzoyl)amino]caprylate is used to form a weak noncovalent association with the polypeptide, wherein the permeation enhancer favors membrane transport and further dissociation once reaching the blood circulation.4878-9453-5582.4Page 16 of 80079065-000004WOPT

[0100] In certain embodiments, the polypeptide is conjugated to oligoarginine, thereby increasing cellular penetration of the peptide into various cell types. Further, in at least one embodiment a noncovalent bond is provided between a polypeptide and a permeation enhancer selected from the group consisting of a cyclodextrin (CD) and a dendrimer, wherein the permeation enhancer reduces peptide aggregation and increasing stability and solubility for the polypeptide.

[0101] In certain embodiments, the dosage form comprises a polypeptide and a transient permeability enhancer (TPE). Permeation enhancers and TPEs may be used to increase orally bioavailability or the peptide. One example of a TPE that may be used is an oily suspension formulation that disperses a powder containing sodium caprylate and polypeptide (Tuvia, S. et al., Pharmaceutical Research, Vol.31, No.8, pp.2010-2021 (2014). Dose and Use

[0102] In various embodiments, a drug load of at least 0.05% by weight for the polypeptide is provided in the enteric coated composition. In some embodiments, the polypeptide is present in about 0.1% to about 0.5%, about 0.5% to about 2%, about 2% to about 5%, about 5% to about 10%, or greater (by weight) in the enteric coated composition. In some embodiments, the polypeptide is present in about 0.1% to about 0.2% (by weight) in the enteric coated composition. In some embodiments, the polypeptide is present in about 0.2% to about 0.3% (by weight) in the enteric coated composition. In some embodiments, the polypeptide is present in about 0.3% to about 0.4% (by weight) in the enteric coated composition. In some embodiments, the polypeptide is present in about 0.4% to about 0.5% (by weight) in the enteric coated composition.

[0103] In some embodiments, the enteric coated composition, or the dosage form, is provided in an amount that accounts for a dose of the polypeptide(s) at 10-50, 50-100, 100-200, 200-300, 300- 400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg / kg of a subject, or 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-50, 50-100 mg / kg of a subject. In some embodiments, a dosage form is provided in an amount that accounts for a dose of the polypeptide(s) at 0.1 – 50 mg. In some embodiments, a dosage form is provided in an amount that accounts for a dose of the polypeptide(s) at 0.25 – 50 mg. In some embodiments, a dosage form is provided in an amount that accounts for a dose of the polypeptide(s) at 0.5 – 20 mg. In some embodiments, a dosage form for administration to a human in a day is provided in an amount that accounts for a dose of the polypeptide(s) at 0.25 – 50 mg / day. In some embodiments, a dosage form for administration to a human in a day is provided in an amount that accounts for a dose of the polypeptide(s) at 0.5 – 20 mg / day. In some embodiments, a dosage form for administration to a 70 kg human in a day is provided in an amount that accounts for a dose of the polypeptide(s) at about 3 mg / day.

[0104] For example, a size 000 gelatin capsule may be filled with about 1 ml (or about 0.8-1.4 ml) volume; and each enteric coated, polypeptide-MCC bead may contain about 1.67 mg (or 1-5 mg) polypeptide per gram of the enteric coated, polypeptide-MCC bead; and so at a bead density of about4878-9453-5582.4Page 17 of 80079065-000004WOPT0.6 g / ml, one size 000 gelatin capsule may be filled with beads that account for about 1 mg (or about 0.5-4 mg considering capsule fill volume range, or about 0.6-3 mg considering polypeptide loading range) of the polypeptide; OR at a bead density of about 0.8 g / ml, one size 000 gelatin capsule may be filled with beads that account for about 1.3 mg (or about 0.64-5.6 mg considering both capsule fill volume range and polypeptide loading range) of the polypeptide; OR at a bead density of about 0.3 g / ml, one size 000 gelatin capsule may be filled with beads that account for about 0.5 mg (or about 0.25-2 mg considering both capsule fill volume range and polypeptide loading range) of the polypeptide; OR at a bead density of about 1.2 g / ml, one size 000 gelatin capsule may be filled with beads that account for about 2 mg (or about 1-8 mg considering both capsule fill volume range and polypeptide loading range) of the polypeptide.

[0105] In some embodiments, a dosage form comprises a gelatin capsule that is filled with enteric coated, polypeptide-embedded beads that account for 0.5-8 mg of the polypeptide. In some embodiments, a dosage form comprises a gelatin capsule that is filled with enteric coated, polypeptide- embedded beads that account for 1-4 mg of the polypeptide. In some embodiments, a dosage form comprises a gelatin capsule that is filled with enteric coated, polypeptide-embedded beads that account for about 2-3.5 mg of the polypeptide. In some embodiments, a dosage form comprises a gelatin capsule that is filled with enteric coated, polypeptide-embedded beads that account for about 3 mg of the polypeptide. In additional embodiments, a dosage form comprises an enteric coated tablet having a compressed formulation of polypeptide-embedded, MCC or other excipients that account for 0.5-8 mg of the polypeptide. In additional embodiments, a dosage form comprises an enteric coated tablet having a compressed formulation of polypeptide-embedded, MCC or other excipients that account for 1-4 mg of the polypeptide. In additional embodiments, a dosage form comprises an enteric coated tablet having a compressed formulation of polypeptide-embedded, MCC or other excipients that account for 2-3.5 mg of the polypeptide. In additional embodiments, a dosage form comprises an enteric coated tablet having a compressed formulation of polypeptide-embedded, MCC or other excipients that account for about 3 mg of the polypeptide.

[0106] In various embodiments, the enteric coated composition, or its dosage form, is administered to a subject with an inflammatory condition in gastrointestinal tract. In other embodiments, the enteric coated composition, or its dosage form, is administered to a subject with acute lung injury or a metabolic disorder.

[0107] Preferably the enteric coated composition, or its dosage form, is administered orally.

[0108] In some embodiments, the enteric coated composition, or its dosage form treats a subject with the inflammatory condition. In some embodiments, the enteric coated composition, or its dosage form treats a subject with the acute lung injury. In some embodiments, the enteric coated composition, or its dosage form treats a subject with the metabolic disorder. In some embodiments, the enteric coated composition, or its dosage form treats a subject with heart failure (HF), including HF4878-9453-5582.4Page 18 of 80079065-000004WOPTwith reduced ejection fraction (HFrEF). In some embodiments, the enteric coated composition, or its dosage form alleviates symptoms of the inflammatory condition. In some embodiments, the enteric coated composition, or its dosage form alleviates symptoms of the acute lung injury. In some embodiments, the enteric coated composition, or its dosage form alleviates symptoms of the metabolic disorder in the subject.

[0109] Inflammatory conditions in gastrointestinal tract include but are not limited to inflammatory bowel disease (ulcerative colitis, Crohn’s disease), necrotizing enterocolitis, radiation- induced gastrointestinal injury. Metabolic disorders include but are not limited to diabetes and non- alcoholic hepatosteatosis (NASH). Preparation

[0110] Figure 16A provides a schematic process of preparing an enteric coated composition disclosed herein. A polypeptide is added during the granulation step, so that the polypeptide, a binding agent, and the MCC are in one solution for granulation. Upon granulation, an enteric coating solution can be spray coated on the granulated core.

[0111] In some embodiments, a method of preparing an enteric coated composition is provided, including mixing the polypeptide, the microcrystalline cellulose, and a binding agent in a slurry; generating particles from the slurry by granulation, extrusion, and / or spheronization; and spraying coating a pH-sensitive polymer on the particles.

[0112] In some embodiments, a method of preparing an enteric coated tablet is provided, including mixing the polypeptide, the microcrystalline cellulose, and a binding agent in a slurry; compressing the mixture on a tablet press to form a compressed formulation; and spray- or dip- coating the compressed formulation with a pH-sensitive polymer. In a further embodiment, the coated tablet may be further compressed into a shape of choice. Polypeptide

[0113] Compounds comprising one or more modifications compared to the amino acid sequence of NRG-4(5-50) (SEQ ID NO:1) are provided, wherein the compounds have one or more activities and properties exhibited by NRG-4(5-50) or NRG-4. Preferably, the compounds have comparable or superior activities and properties compared to human NRG-4. Modifications described herein include deletion of amino acids (e.g., truncation at N-terminus or C-terminus), substitution of amino acids, addition of amino acids, lipid moieties, or polymers other than an amino acid polymer, and optionally branching in the polypeptide. These compounds with the modifications compared to the amino acid sequence of NRG-4(5-50) are also called analogs of NRG-4 or variants of NRG-4.

[0114] Some embodiments provide a compound, or a polypeptide, comprising Formula I. Formula I:4878-9453-5582.4Page 19 of 80079065-000004WOPTXaa0- [Xaa1Xaa2Xaa3Xaa4HXaa6Xaa7PCGPSHXaa14SXaa16CXaa18Xaa19Xaa20GLCYVIPTXaa29PXaa31PFC RCXaa37ENYTGARCXaa46Xaa47Xaa48Xaa49L]-Xaaμ(Xaa0-[SEQ ID NO:42]-Xaaμ), wherein Xaa1is absent; Xaa2is absent or K; Xaa3is absent or G or T or S; Xaa4is absent or D or E or N or Q; Xaa6is E or D; Xaa7is E or Q; Xaa14is K or R; Xaa16is F or Y; Xaa18is L, I, or V; Xaa19is N, Q, K, R or H; Xaa20is G, A, N, Q, S, E, or D; Xaa29is I or L; Xaa31is S or G; Xaa37is V or I; Xaa46Xaa47is EE, DQ, DN, or EQ; Xaa48Xaa49is VF or FL; Xaa0is absent or selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:47), and (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is S, Q, A, or E and n=1-5, and Xaa0is optionally further modified with a first hydrophobic moiety; and Xaaμis absent, or selected from the group consisting of: PGS, S, GS, GGS, GGGS (SEQ ID NO:47), (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is S, Q, A, or E and n=1-5, Pro^Gly^Ser^Lys J-Glu ([SEQ ID NO:44] J-Glu), Ser^Lys J-Glu, Gly^Ser^Lys J-Glu, Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:45] J-Glu), Gly^Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:46] J-Glu), and a combination of (GGGGXaa^)n(SEQ ID NO:43) and Lys J-Glu, wherein the bonds depicted by represent an amide linkage between İ-amino group of the Lys and Ȗ-carboxyl group of the Glu, optionally said Glu being further bound to a second hydrophobic moiety, wherein if Xaa46Xaa47is EE, then Xaa48Xaa49is VF, wherein if Xaa46Xaa47is DQ, then Xaa48Xaa49is VF or FL, wherein if Xaa46Xaa47is DN, then Xaa48Xaa49is VF, and wherein if Xaa46Xaa47is EQ, then Xaa48Xaa49is VF, and wherein if Xaa19is H, then Xaa47is not Q,4878-9453-5582.4Page 20 of 80079065-000004WOPTwherein if Xaa0is absent, the N-terminus the polypeptide is optionally modified with a hydrophobic moiety, and wherein if Xaaμis absent, the C-terminus the polypeptide is optionally modified with a hydrophobic moiety.

[0115] In some embodiments, the polypeptide consists of Formula I.

[0116] In some aspects, only one of the first hydrophobic moiety and the second hydrophobic moiety is present in SEQ ID NO:42. In some aspects, both Xaa0and Xaaμare absent.

[0117] In some embodiments, if Xaa0and Xaa1are absent, if Xaa2is K, and the N-terminus is modified with a hydrophobic moiety, then K is modified to Lys(AEEAc-AEEAc-Ȗ-Glu- C=O(CH2)nR, where n can be an integer from 12-22 and R is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, - CO2N(CH3)2, -CH3, or -NH2; that is, the İ-amino group of Lys (i.e., the Lys side chain) is substituted with an amide bonded, first 8-amino-3,6-dioxa-octanoic acid (AEEAc) moiety, (hence an amide bond is between the Lys’ İ-amino group and the carboxyl group of the first AEEAc,) and the amino group of the first AEEAc moiety is substituted with an amide bonded, second AEEAc moiety (hence an amide bond is between the amino group of the first AEEAc and the carboxyl group of the second AEEAc,) and the amino group of the second AEEAc moiety is substituted with an amide bonded, Glu- C=O(CH2)nR moiety at the gamma-carboxyl group of the glutamic acid, (hence an amide bond is between the amino group of the second AEEAc and the gamma-carboxyl group of the glutamic acid).

[0118] In some embodiments, if Xaa0, Xaa1, Xaa2are absent, if Xaa3is G, and the N-terminus is modified with a hydrophobic moiety, then G is modified with the hydrophobic moiety C=O(CH2)nR, where n can be an integer from 12-22 and R is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, - CO2N(CH3)2, -CH3, or -NH2.

[0119] Embodiments relating to the hydrophobic moiety at the N-terminus, C-terminus, or both are discussed in further detail herein.

[0120] Some embodiments provide a compound, or a polypeptide, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:42, i.e., a variant of SEQ ID NO:42. Preferably, the compound or polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:42 has comparable or superior activities or properties compared to SEQ ID NO:42, compared to human NRG-4, or compared to a fragment (residues 5-50) of human NRG-4 (SEQ ID NO:1).

[0121] Some embodiments provide a compound, or a polypeptide, comprising or consisting of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Formula I, i.e., a variant of Formula I. Preferably, the compound or polypeptide having at4878-9453-5582.4Page 21 of 80079065-000004WOPTleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Formula I has comparable or superior activities or properties compared to Formula I, compared to human NRG-4, or compared to a fragment (residues 5-50) of human NRG-4 (SEQ ID NO:1).

[0122] In various aspects the compound or the polypeptide is not human NRG-4 (5-50), human NRG-4(1-62), epidermal growth factor (EGF), human NRG-3, rat NRG-4, mouse NRG-4, gorilla NRG- 4, or chimpanzee NRG-4. In various aspects the compound or polypeptide is not an NRG-4 analog described in U.S. Patent Nos.6,544,759 or 7,094,882, herein incorporated by reference in their entirety as though fully set forth.

[0123] In some embodiments, activities exhibited by a polypeptide selected from Formula I, SEQ ID NO:42, SEQ ID NO:1, or human NRG-4 include: inducing homodimerization of ErbB4 subunits, having specificity in inducing homodimerization of ErbB4 subunits over homodimerization of EGFR subunits (e.g., by a difference in EC50 of at least 1,000 fold), having stability with respect to resisting enzymatic degradation in intestinal fluid or a simulated intestinal fluid, and having stability with respect to resisting enzymatic degradation in gastric fluid or a simulated gastric fluid. In some aspects, the capability to induce homodimerization of ErbB4 subunits, or heterodimerization of other ERBB family subunits, can be verified by using PATHHUNTER® Dimerization assays available from Eurofins DiscoverX Corporation, Fremont, CA. In some aspects, the polypeptide analogs of NRG-4 as disclosed herein have at least comparable (e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), or superior, stability in resisting enzymatic degradation (such as cleavage by chymotrypsin, trypsin, or pepsin in acidic environment mimicking gastric or intestinal conditions), compared to NRG-4, NRG-4(5-50), or SEQ ID NO:42, or Formula I.

[0124] In certain embodiments, the polypeptide disclosed herein include structural motifs of component peptides that impart a desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristic to the peptide, and structural motifs of analogs or derivatives of component peptides that impart a desired chemical stability, conformational stability, metabolic stability, bioavailability, organ / tissue targeting, receptor interaction, protease inhibition, plasma protein binding, or other pharmacokinetic characteristic to the polypeptide.

[0125] In one embodiment, the polypeptide includes a heterologous C-terminal tail or N- terminal extension. In another aspect, the polypeptide comprises at least one modification, substitution, deletion or addition that provides one or more enhanced properties, e.g. increased resistance to proteolytic digestion (thus prolonging half-life), fatty acyl derivatization that facilitates albumin binding to reduce renal clearance or can facilitate association with fatty acid transporters on the cell surface of enterocytes, leading to greater residence time in the gastrointestinal (GI) tract.4878-9453-5582.4Page 22 of 80079065-000004WOPT

[0126] Various embodiments provide that the compound or polypeptide further includes a hydrophobic moiety covalently linked to the N-terminus, the C-terminus, or both.

[0127] In some embodiments, the hydrophobic moiety is a lipid moiety. Lipid moieties useful in the present invention include from 1 to 30-carbon long acyl chains, from 1 to 10 acyl chains, or from 1 to 6 acyl chains, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 acyl chains. The lipid moieties can be prepared from fatty acids, which include, but are not limited to, capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), isostearic acid (C18), oleic acid (C18), vaccenic acid (C18), linoleic acid (C18), alpha-linoleic acid (C18), gamma-linolenic acid (C18), arachidic acid (C20), gadoleic acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20), behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22), lignoceric acid (C24) and hexacosanoic acid (C26). For example, the carboxylic acid group of a C10-30fatty acid is reacted with an amine on the N-terminus residue of a polypeptide or on the side chain of an amino acid residue in the polypeptide, thereby forming an amide linkage, resulting in a conjugation between the fatty acid and the polypeptide, with an exemplary structure of C10-30acyl-polypeptide. Exemplary acyl groups in the lipid moieties include C10-20acyl chains, such as C10, C12, C14, C16, C18, or C20acyl groups. In some embodiments, the lipid moieties have at least one C14 acyl group, or at least one C16acyl group. When the lipid moieties include more than one acyl group, the lipid moiety also includes a branched linker providing for attachment of multiple acyl groups. The branched linkers useful in the present invention include, but are not limited to, lysine, glutamic acid and other branched amines and carboxylic acids. In some embodiments, the lipid moiety includes from one to six C10-20acyl groups. The lipid moiety can include one, two, three, four, five, or six C10-20acyl groups. In some embodiments, the lipid moiety includes one, two or four C10-20acyl groups. In some embodiments, the lipid moiety includes one C10-20acyl group. In some embodiments, the lipid moiety includes two C10-20acyl groups. The hydrophobic moiety of C10-30acyl group can also be phrased as a half-life extension moiety, with illustrative structures: ,,4878-9453-5582.4Page 23 of 80079065-000004WOPT.

[0128] In some embodiments, the hydrophobic moiety is HO2CíRíC(=O)-, wherein R can be aliphatic or aromatic alkane / alkene / alkyne. That is, a polypeptide is modified with a hydrophobic moiety by reacting the polypeptide with a C3-C30 dicarboxylic acid (di-acid). The hydrophobic moiety can be prepared from dicarboxylic acids, which include but are not limited to malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (or tridecanedioic acid, C13), tetradecanedioic acid (C14), pentadecanedioic acid (C15), thapsic acid (or hexadecanedioic acid, C16), heneicosadioic acid (C21), docosanedioic acid (C22), and triacontanedioic acid (C30). In some aspects, the dicarboxylic acid is a straight saturated medium chain length (C9-C12) dicarboxylic acid. In other aspects, the dicarboxylic acid can be a branched dicarboxylic acid. In some embodiments, a polypeptide is modified at N-terminus with a hexadecanedioic acid or a tetradecanedioic acid. For example, one of the carboxylic acid groups of a di-acid is reacted with an amine on the N-terminus residue or on the side chain of an amino acid residue in the polypeptide, thereby forming an amide linkage, resulting in a conjugation between the di-acid and the polypeptide.

[0129] In some embodiments, the hydrophobic moiety is C=O(CH2)nR, where n can be an integer from 12-22 and R is -CO2H, -CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2.

[0130] The hydrophobic moiety derived from C10-30di-acids can also be phrased as a half-life extension moiety, with illustrative structures: , ,,4878-9453-5582.4Page 24 of 80079065-000004WOPT, .

[0131] In certain embodiments, a half-life extension moiety is bound directly to a peptide, while in other embodiments, a half-life extension moiety is bound to the peptide via a linker moiety. In some embodiments the linker is AEEAc-AEEAc (AEEAc = (2-[2-(2-Aminoethoxy)ethoxy]acetic acid, or also known as 8-Amino-3,6-dioxaoctanoic acid).

[0132] In other embodiments, the hydrophobic moiety is a polymer other than an amino acid polymer. Polymers useful as the hydrophobic moiety include hydrophobic polymers such as polybutadiene, polystyrene, polyacrylates, polymethacrylates, polydiacetylene, and the like. In some embodiments, the polymer can be about 250 Da, or about 300 Da, or about 350 Da, or about 400 Da, or about 500 Da, or about 1000 Da, or about 1250 Da, or about 1500 Da, or about 1750 Da, or about 2000 Da, or about 3000 Da, or about 4000 Da, or about 5000 Da.

[0133] In other embodiments, the compound or polypeptide further includes a C-terminal modification, such as amides (NH2), N-alkyl amides, aldehydes, esters, p-Nitroanilide (pNA), or 7- amino-4-metylcoumarin (AMC). In other embodiments, the compound or polypeptide further includes a N-terminal modification, such as acetyl, formyl, pyroglutamyl (pGlu), urea, or carbamate.

[0134] Further embodiments provide that the compound or polypeptide further includes a linker between the hydrophobic moiety and the terminal amino acid residue of SEQ ID NO:42. Further embodiments provide that the compound or polypeptide further includes a linker between the hydrophobic moiety and the terminal amino acid residue of Formula I.

[0135] In some embodiments, the linker is a short peptide, such as a dimer of two amino acids, a tri-mer of three amino acids, or a peptide selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:47), and (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is Q, A, E or S and n=1-5. In some embodiments the linker is AEEAc-AEEAc.

[0136] As such, various embodiment provide a compound or polypeptide comprising a N- terminus modification compared to SEQ ID NO:42, by the formula: (Formula Ia): Xaa0- [Xaa1Xaa2Xaa3Xaa4HXaa6Xaa7PCGPSHXaa14SXaa16CXaa18Xaa19Xaa20GLCYVIPTXaa29PXaa31PFC RCXaa37ENYTGARCXaa46Xaa47Xaa48Xaa49L] (Xaa0-[SEQ ID NO:42]), wherein Xaa0is selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:47), and (GGGGXaa^)n(SEQ ID NO:43), wherein Xaa^is S, Q, A, or E and n=1-5, and4878-9453-5582.4Page 25 of 80079065-000004WOPTXaa0is optionally further modified with a hydrophobic moiety (such as a lipid moiety, a C10 to C30 acyl, or HO2Cí(CH2)níC(=O)-, wherein n is an integer from 12 -22); wherein Xaa1is absent; Xaa2is absent or K; Xaa3is absent or G or T or S; Xaa4is absent or D or E or N or Q; Xaa6is E or D; Xaa7is E or Q; Xaa14is K or R; Xaa16is F or Y; Xaa18is L, I, or V; Xaa19is N, Q, K, R or H; Xaa20is G, A, N, Q, S, E, or D; Xaa29is I or L; Xaa31is S or G; Xaa37is V or I; Xaa46Xaa47is EE, DQ, DN, or EQ; Xaa48Xaa49is VF or FL; and wherein if Xaa46Xaa47is EE, then Xaa48Xaa49is VF, wherein if Xaa46Xaa47is DQ, then Xaa48Xaa49is VF or FL, wherein if Xaa46Xaa47is DN, then Xaa48Xaa49is VF, and wherein if Xaa46Xaa47is EQ, then Xaa48Xaa49is VF, and wherein if Xaa19is H, then Xaa47is not Q.

[0137] In some embodiments, the polypeptide consists of Formula Ia.

[0138] In various aspects the compound or the polypeptide is not human NRG-4 (5-50), human NRG-4(1-62), EGF, human NRG-3, rat NRG-4, mouse NRG-4, gorilla NRG-4, or chimpanzee NRG- 4. In various aspects the compound or polypeptide is not an NRG-4 analog described in U.S. Patent Nos.6,544,759 or 7,094,882, herein incorporated by reference in their entirety as though fully set forth.

[0139] Additional embodiments provide a compound or polypeptide comprising a C-terminus modification compared to SEQ ID NO:42, by the formula: (Formula Ib) Xaa1Xaa2Xaa3Xaa4HXaa6Xaa7PCGPSHXaa14SXaa16CXaa18Xaa19Xaa20GLCYVIPTXaa29PXa a31PFCRCXaa37ENYTGARCXaa46Xaa47Xaa48Xaa49LXaaμ([SEQ ID NO:42]-Xaaμ), wherein Xaaμis selected from PGS, S, GS, GGS, GGGS (SEQ ID NO:47), (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is S, Q, A, or E and n=1-5, Pro^Gly^Ser^Lys J-Glu ([SEQ ID NO:44]4878-9453-5582.4Page 26 of 80079065-000004WOPTJ-Glu) wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, Ser^Lys J-Glu (SEQ ID NO: 52) wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, Gly^Ser^Lys J-Glu wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:45] J-Glu) wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, Gly^Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:46] J-Glu) wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, or a combination of (GGGGXaa^)n(SEQ ID NO:43) and Lys J-Glu wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and (optionally further bound) to the carboxyl group of a fatty acid (or a carboxyl group of a dicarboxylic acid) by its amino group, and wherein Xaaμis optionally further modified with a hydrophobic moiety (such as a lipid moiety, a C10-30acyl, or HO2CíRíC(=O)-, wherein R can be aliphatic or aromatic) when the glutamic acid in any of SEQ ID NOs: 46-48, if present, is not already bound to a hydrophobic moiety; and wherein Xaa1is absent; Xaa2is absent or K; Xaa3is absent or G or T or S; Xaa4is absent or D or E or N or Q; Xaa6is E or D; Xaa7is E or Q; Xaa14is K or R; Xaa16is F or Y; Xaa18is L, I, or V; Xaa19is N, Q, K, R or H; Xaa20is G, A, N, Q, S, E, or D; Xaa29is I or L; Xaa31is S or G; Xaa37is V or I; Xaa46Xaa47is EE, DQ, DN, or EQ; Xaa48Xaa49is VF or FL;4878-9453-5582.4Page 27 of 80079065-000004WOPTwherein the bonds depicted by represent an amide linkage between İ-amino group of the Lys and Ȗ-carboxyl group of the Glu, optionally said Glu being further bound to a second hydrophobic moiety, wherein if Xaa46Xaa47is EE, then Xaa48Xaa49is VF, wherein if Xaa46Xaa47is DQ, then Xaa48Xaa49is VF or FL, wherein if Xaa46Xaa47is DN, then Xaa48Xaa49is VF, and wherein if Xaa46Xaa47is EQ, then Xaa48Xaa49is VF, and wherein if Xaa19is H, then Xaa47is not Q. Note: the bonds between lysine and another amino acid depicted by ^^represent an amide linkage between the terminal amine of the lysine and the carboxyl end of the bonded amino acid, wherein the bonds depicted by represent an amide linkage between the side chain amine (i.e., İ- amino group) of the lysine and, in this case, the gamma-carboxyl group of glutamic acid, or if other cases than J-Glu, the carboxyl end of the bonded amino acid.

[0140] In some embodiments, the polypeptide consists of Formula Ib.

[0141] In various aspects the compound or the polypeptide is not human NRG-4 (5-50), human NRG-4(1-62), EGF, human NRG-3, rat NRG-4, mouse NRG-4, gorilla NRG-4, or chimpanzee NRG- 4. In various aspects the compound or polypeptide is not an NRG-4 analog described in U.S. Patent Nos.6,544,759 or 7,094,882, herein incorporated by reference in their entirety as though fully set forth.

[0142] Yet in some embodiments, the compound or polypeptide, e.g., SEQ ID NO:42, a variant of SEQ ID NO:42, a variant of SEQ ID NO:1, Formula I, Formula Ia, Formula Ib, variant of variant of Formula I, variant of variant of Formula Ia, OR variant of variant of Formula Ib does not comprise a hydrophobic moiety on its terminus.

[0143] Typically, a polypeptide analog of NRG-4 disclosed herein is sequence aligned with NRG-4 or NRG-4(5-50) for comparing and detecting similarities (and dissimilarities) between peptide / protein sequences. Sequence alignment tools are known and available such as Protein BLAST. In various implementations, the disulfide-forming cysteine residues of the polypeptide analog of NRG- 4 disclosed herein are lined up for homology, or correspond to, the disulfide-forming cysteine residues of NRG-4 or NRG-4(5-50), as shown in Table 4.

[0144] In various embodiments, a compound or polypeptide disclosed herein is a variant of Formula I, Formula Ia or Formula Ib, and the variant does not necessarily have the same number of amino acids as Formula I, Formula Ia or Formula Ib, respectively, so dissimilarities compared to Formula I, Formula Ia or Formula Ib, respectively are described in terms of the corresponding residue position in SEQ ID NO:42. In some aspects, the variant has a longer length, i.e., more amino acids, compared to SEQ ID NO:1 or Formula I, Formula Ia or Formula Ib, respectively. In other aspects, the variant has a shorter length, i.e., fewer amino acids, compared to SEQ ID NO:1 or Formula I, Formula Ia or Formula Ib, respectively.4878-9453-5582.4Page 28 of 80079065-000004WOPT

[0145] In various embodiments, a compound or polypeptide disclosed herein is a variant of SEQ ID NO:42, and the variant does not necessarily have the same number of amino acids as SEQ ID NO:42, so dissimilarities compared to SEQ ID NO:42 are described in terms of the corresponding residue position in SEQ ID NO:42. In other embodiments, a compound or polypeptide disclosed herein is a variant of SEQ ID NO:1, and the variant does not necessarily have the same number of amino acids as SEQ ID NO:1, so dissimilarities compared to SEQ ID NO:1 are described in terms of the corresponding residue position in SEQ ID NO:1. See aligned sequences shown in Table 4. In some aspects, the variant has a longer length, i.e., more amino acids, compared to SEQ ID NO:1 or SEQ ID NO:42. For example, the variant can have 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 amino acids in size. In other aspects, the variant has a shorter length, i.e., fewer amino acids, compared to SEQ ID NO:1 or SEQ ID NO:42. For example, the variant has 45, 44, 43, 42, 41, or 40 amino acids in size.

[0146] In various embodiments, the variant has residues of Cys, in positions corresponding to the Xaa9and the Xaa23of SEQ ID NO:42, capable of forming a first disulfide bond; residues of Cys, in positions corresponding to the Xaa17and the Xaa34of SEQ ID NO:42, capable of forming a second disulfide bond; and residues of Cys, in positions corresponding to the Xaa36and the Xaa45of SEQ ID NO:42, capable of forming a third disulfide bond.

[0147] In some embodiments, the variant is a polypeptide comprising a fragment that comprises three amino acids corresponding in sequence alignment to the Xaa48through the Xaa50of SEQ ID NO:42. In other embodiments, the variant contains a sufficient number of amino acids corresponding to the Xaa5through the Xaa50of SEQ ID NO:42 when sequence aligned with SEQ ID NO:42. That is, the C-terminus fragment of SEQ ID NO:42, such as Xaa46through Xaa50, is not truncated in formulating a variant.

[0148] In further embodiments, the variant’s residue corresponding to the Xaa50of SEQ ID NO:42 is not Pro (P). For example, SEQ ID NO:4 having a Pro at position corresponding to the Xaa50of SEQ ID NO:42 has a loss or significant reduction in activity in inducing homodimerization of ErbB4 subunits compared to NRG-4.

[0149] In further embodiments, the variant’s residue corresponding to the Xaa24of SEQ ID NO:42 is not Gln (Q) or Val (V). For example, SEQ ID NOs:5 and 6 having a Q or V at position corresponding to the Xaa24of SEQ ID NO:42 has a loss or significant reduction in activity in inducing homodimerization of ErbB4 subunits compared to NRG-4.

[0150] In further embodiments, the variant’s residue corresponding to the Xaa50of SEQ ID NO:42 is Leu (L) if residues corresponding to the Xaa46through the Xaa49of SEQ ID NO:42 are Asp- Gln-Val-Phe (SEQ ID NO:37). For example, SEQ ID NOs: 15 and 16.

[0151] In further embodiments, the variant’s residues corresponding to the Xaa46through the Xaa48of SEQ ID NO:42 are not Asp-Gln-Phe if the variant’s C-terminus is not further modified. For4878-9453-5582.4Page 29 of 80079065-000004WOPTexample, SEQ ID NO:23 having Asp-Gln-Phe as the C-terminus fragment has a significant reduction in activity in inducing homodimerization of ErbB4 subunits compared to NRG-4.

[0152] In other embodiments, a variant can have C-terminal tail of Asp-Gln-Phe, as long as the C-terminus residue is not further modified.

[0153] In further embodiments, a variant’s residue corresponding to the Xaa49of SEQ ID NO:42 is not a D-enantiomeric amino acid.

[0154] In further embodiment, the variant does not comprise a hydrophobic moiety covalently linked to the N-terminus of the polypeptide, if residues corresponding to the Xaa46through the Xaa50of SEQ ID NO:42 are Asp-Gln-Val-Phe-Leu (SEQ ID NO:41) and a residue corresponding to the Xaa19of SEQ ID NO:42 is His. For example, SEQ ID NO:26 having residues corresponding to the Xaa46Xaa47Xaa48Xaa49Xaa50of SEQ ID NO:42 as Asp46-Gln47-Val48-Phe49-Leu50(SEQ ID NO:41), H at Xaa19and having a lipid moiety on the N-terminus, has a significant reduction in activity in inducing homodimerization of ErbB4 subunits compared to NRG-4.

[0155] In some embodiments, a compound or polypeptide is SEQ ID NO:7. In some embodiments, a compound or polypeptide is SEQ ID NO:8. In some embodiments, a compound or polypeptide is SEQ ID NO:9. In some embodiments, a compound or polypeptide is SEQ ID NO:10. In some embodiments, a compound or polypeptide is SEQ ID NO:15. In some embodiments, a compound or polypeptide is SEQ ID NO:16. In some embodiments, a compound or polypeptide is SEQ ID NO:17. In some embodiments, a compound or polypeptide is SEQ ID NO:21. In some embodiments, a compound or polypeptide is SEQ ID NO:22. In some embodiments, a compound or polypeptide is SEQ ID NO:49. In some embodiments, a compound or polypeptide is SEQ ID NO:50. In some embodiments, a compound or polypeptide is SEQ ID NO:51. In some embodiments, a compound or polypeptide is SEQ ID NO:52. In some embodiments, a compound or polypeptide is SEQ ID NO:53. In some embodiments, a compound or polypeptide is SEQ ID NO:52. In some embodiments, a compound or polypeptide contains a mixture of two or more of SEQ ID NOs:7-10, 15-17, 21, 22, 49, 50, 51, 52, 53 or 54.

[0156] Table of Peptides4878-9453-5582.4Page 30 of 80079065-000004WOPTSynthesis

[0157] The polypeptide disclosed herein may be prepared using standard recombinant techniques or solid phase peptide synthesis (SPPS) techniques known in the art, e.g., using an automated or semi-automated peptide synthesizer, or both.

[0158] The polypeptide disclosed herein can be synthesized in solution or on a solid support in accordance with conventional techniques. Such methods are described, for example, herein and in U.S. Pat. No. 6,610,824 and U.S. Pat. No. 5,686,411 and in patent application Ser. No. 454,533 (filed Dec. 6, 1999), the entirety of which are incorporated herein by reference. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, e.g.,4878-9453-5582.4Page 31 of 80079065-000004WOPTStewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam et al, J. Am. Chem. Soc.105: 6442 (1983); Merrifield, Science 232: 341-7 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, 1-284 (1979).

[0159] Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP / HOBt system and tBoc or Fmoc chemistry (see, Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with capping. Peptides may also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).

[0160] Peptides analogs can be synthesized on a Pioneer continuous flow peptide synthesizer (Applied Biosystems) using PAL-PEG-PS resin (Applied Biosystems) with a loading of 0.2 mmol / g (0.25 mmole scale). Fmoc amino acid (4.0 eq, 1.0 mmol) residues are activated using 4.0 eq HBTU, 4.0 eq of HOBT, 8.0 eq DIEA and coupled to the resin for 1 hour. The Fmoc group is removed by treatment with 20% (v / v) piperidine in dimethylformamide. Final deprotection and cleavage of the peptide from the solid support is performed by treatment of the resin with reagent B (93% TFA, 3% phenol, 3% water and 1% triisopropylsilane) for 2-3 hours. The cleaved peptide is precipitated using tert-butyl methyl ether, and pelleted by centrifugation and lyophilized. Alternately, fully protected peptidyl-Resin is cleaved by E Solution (e.g., TFA : Thioanisole: EDT : Phenol : H2O =87.5 : 5 : 2.5 : 2.5 : 2.5) for about 3 hours, filtered and the filtrate is precipitated in cool ether. Crude product is collected and washed with ether, and dried under vacuum.

[0161] Alternatively, peptides are assembled on a Symphony peptide synthesizer (Protein Technologies, Inc., Woburn, Mass.) using Wang or Rink amide resin (Novabiochem, San Diego, Calif.) with a loading of 0.43-0.49 mmol / g at 0.050-0.100 mmol. Fmoc amino acid (Applied Biosystems, Inc. 5.0 eq, 0.250-0.500 mmol) residues are dissolved at a concentration of 0.10 M in 1-methyl-2- pyrrolidinone. All other reagents (HBTU, HOBT and N,N-diisopropylethylamine) are prepared as 0.55 M dimethylformamide solutions. The Fmoc protected amino acids are then coupled to the resin-bound amino acid using, HBTU (2.0 eq, 0.100-0.200 mmol), HOBT (1.8 eq, 0.090-0.18 mmol), N,N- diisopropylethylamine (2.4 eq, 0.120-0.240 mmol) for 2 hours. Following the last amino acid coupling, the peptide is deprotected using 20% (v / v) piperidine in dimethylformamide for 1 hour. Once peptide sequence is completed, the Symphony peptide synthesizer is programmed to cleave the resin. Trifluoroacetic acid (TFA) cleavage of the peptide from resin is carried out using a reagent mixture composed of 93% TFA, 3% phenol, 3% water and 1% triisopropylsilane. The cleaved peptide is precipitated using tert-butyl methyl ether, pelleted by centrifugation and lyophilized. Alternately, fully protected peptidyl-Resin is cleaved by E Solution (e.g., TFA : Thioanisole: EDT : Phenol : H2O =87.5 : 5 : 2.5 : 2.5 : 2.5) for about 3 hours, filtered and the filtrate is precipitated in cool ether. Crude product is collected and washed with ether, and dried under vacuum.4878-9453-5582.4Page 32 of 80079065-000004WOPT

[0162] Polypeptides can be synthesized by convergent methods such as “native chemical ligation”, and variations thereof, in which two or more peptide fragments with appropriate orthogonally reactive ends are ligated with native amide bond formation. The newly formed peptide can be further ligated to create even longer polypeptides. The individual starting peptides can be derivatized as desired or can be derivatized after a ligation step.

[0163] For refolding, the crude peptides can be dissolved in water and can be oxidized using glutathione in acetonitrile / water to form the disulfide linkages.

[0164] A particularly efficient method of purifying peptides is reverse phase high-performance liquid chromatography (RP-HPLC), followed by characterization of purified product by liquid chromatography / mass spectrometry (LC / MS). Additional confirmation of purity is obtained by determining amino acid analysis. The folded peptides can be purified by RP-HPLC using, e.g., a Waters Delta Prep 3000 system and a C4, C8, or C18 preparative column (10^, 2.2×25 cm; Vydac, Hesperia, Calif.) and an acetonitrile / water gradient containing 0.1% TFA.

[0165] In various embodiments, the polypeptides of the invention are isolated polypeptides. Generally, “purified” will refer to a peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the peptides in the composition.

[0166] The purified NRG-4 analogs described below were analyzed by analytical RP-HPLC to assess purity of peptide and identity was confirmed by LC / MS and amino acid content. The analogs were demonstrated to be >90% pure by RP-HPLC and mass results all agreed with calculated values.

[0167] In some embodiments of synthesizing a polypeptide (such as polypeptides of SEQ ID NOs:9, 10, 28 and 29 or SEQ ID NO:43) whose C terminal tail is Lys J-Glu, wherein Glu (E) is bound to the İ-amino group of Lys (K) by its Ȗ-carboxyl group and further bound by its amino group to the carboxyl group of a fatty acid or to a carboxyl group of a dicarboxylic acid, the process includes: Lys is the first amino acid on the resin, and at some point during the synthesis of the polypeptide, the H-amine of Lys reacts with a modified glutamic acid to form the amide linkage, wherein the modified glutamic acid is (1) activated in its J-carboxyl group by N-hydroxysuccinimide (NHS) (forming J- C(=O)-NHS ester) for bonding with the H-amine of Lys, (2) protected in its Į-carboxyl group by tert- butyl protecting group, and (3) bound to a fatty acid or a di-acid by its (one and only) Į-amino group. At the end of the synthesis, the tert-butyl protecting group is removed to give the C-terminal tail. An exemplary modified glutamic acid having (1), (2), and (3) being palmitoyl modified, is shown below:4878-9453-5582.4Page 33 of 80079065-000004WOPTMethods of using the compositions

[0168] Various embodiments provide for a method for treating a subject with an inflammatory condition in gastrointestinal tract (GI), comprising administering a pharmaceutical composition of the present invention as described herein to the subject.

[0169] In various embodiments, the method comprising orally administering the pharmaceutical composition and releasing the polypeptide in the subject’s colon.

[0170] In various embodiments, the inflammatory condition comprises ulcerative colitis, Crohn’s disease, necrotizing enterocolitis, or radiation-induced gastrointestinal injury.

[0171] Further embodiments provide for a method for treating a subject with a GI-associated injury, comprising administering a pharmaceutical composition of the present invention as described herein to the subject. Exemplary GI-associated injuries include inflammation injury such as having an overexpression of TNF / IFNy, with GI symptoms / signs such as intestinal inflammation and / or colonic cell apoptosis.

[0172] Various embodiments provide for a method for treating a subject with acute lung injury or a metabolic disorder, comprising administering a pharmaceutical composition of the present invention to the subject.

[0173] In various embodiments, the metabolic disorder comprises diabetes or non-alcoholic steatohepatitis.

[0174] In various embodiments, the pharmaceutical composition further comprises a permeation enhancer, and the administration is a systemic administration.

[0175] In some embodiments, a method of treating a subject with an inflammatory condition in the gastrointestinal (GI) tract, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the inflammatory condition, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein. In various embodiments, treatment, inhibition or reduction of symptoms of IBD in a subject may prevent, reduce the likelihood of having and / or treat colorectal cancer.

[0176] In some embodiments, a method of improving stool form in a subject with an inflammatory condition in the gastrointestinal (GI) tract, includes administering to the subject a4878-9453-5582.4Page 34 of 80079065-000004WOPTtherapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0177] In some embodiments, a method of reducing blood in stool in a subject with an inflammatory condition in the gastrointestinal (GI) tract, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0178] In some embodiments, a method of reducing weight loss in a subject with an inflammatory condition (e.g., in the GI tract, colitis, enterocolitis, etc. as described herein), includes administering to the subject a composition of the present invention comprising any one or more of the polypeptides described herein.

[0179] In some embodiments, a method of treating a subject with lung injury, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the lung injury, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0180] In some embodiments, a method of treating a subject with diabetes, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the diabetes, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0181] In some embodiments, a method of treating a subject with nonalcoholic steatohepatitis (NASH), inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the NASH, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein. In some embodiments, a method of treating a subject with nonalcoholic fatty liver disease (NAFLD), inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the NAFLD, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein. In some embodiments, a method of treating a subject with liver cancer, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the liver cancer, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0182] In some embodiments, a method of treating a subject with acute lung injury, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the acute lung injury, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0183] In some embodiments, a method of treating a subject with necrotizing enterocolitis, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the necrotizing4878-9453-5582.4Page 35 of 80079065-000004WOPTenterocolitis, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0184] In some embodiments, a method of treating a subject having undergone radiation or in need of radiation, or inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the radiation-induced GI injury, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein.

[0185] In some embodiments, a method of treating a subject with a cardiovascular indication such as endothelial inflammation and / or atherosclerosis, inhibiting or reducing severity or symptoms of, or promoting prophylaxis of the cardiovascular indication, includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein. In some embodiments, a method of treating a subject with heart failure (HF), including HF with reduced ejection fraction (HFrEF), inhibiting or reducing severity or symptoms of, or promoting prophylaxis of heart failure (HF), including HF with reduced ejection fraction (HFrEF), includes administering to the subject a therapeutically effective amount of a composition of the present invention comprising any one or more of the polypeptides described herein. In an embodiment, treatment, inhibition or reduction of symptoms of heart failure (HF), including HF with reduced ejection fraction (HFrEF)in a subject may prevent and / or treat heart failure (HF), including HF with reduced ejection fraction (HFrEF).

[0186] Various embodiments provide methods for inhibiting inflammation-associated death of small intestinal and colonic epithelial cells in a subject in need thereof, by administering a composition of the present invention comprising any one or more of the polypeptides described herein to the subject. Some embodiments provide methods for improving healing of gut mucosa in a subject with an inflammatory condition in the gut, by administering to the subject a composition of the present invention comprising any one or more of the polypeptides described herein.

[0187] Further embodiments provide methods for treating an IBD, a colitis, or radiation- induced gastrointestinal injury in a subject detected with an increased number of ErbB4+ pro- inflammatory macrophages in at least a portion of the GI tract of the subject relative to a reference value, by administering a composition of the present invention comprising any one or more of the polypeptides described herein to the subject detected with the increased number of the ErbB4+ pro- inflammatory macrophages. In some aspects, the reference value is the mean or median number of ErbB4+ macrophages in that portion of the GI tract of a normal subject or a subject that has been diagnosed and successfully treated for the IBD, the colitis, or the radiation-induced gastrointestinal injury.

[0188] Some embodiments provide the methods for treating a disease or condition noted herein in a human include administering to the human a composition of the present invention that account for 0.25 mg – 50 mg of the polypeptide per day. Some embodiments provide the methods for treating a4878-9453-5582.4Page 36 of 80079065-000004WOPTdisease or condition noted herein in a human include administering to the human a composition of the present invention that account for 0.5 mg – 20 mg of the polypeptide per day. In some embodiments, the composition of the present invention is provided in 1, 2, 3 or 4 doses per day. In some embodiments, the composition of the present invention is used for chronic treatment. In some aspects, chronic treatment is a treatment that lasts for about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, or more than 180 days. In other aspects, chronic treatment includes cycles of treatment; for example, administering treatment for a certain number of days and then a period time of no treatment. EXAMPLES

[0189] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. Example 1 Structure Activity Relationship (SAR) Studies

[0190] Table 1 summarizes the polypeptides in the SAR study. The peptides were obtained at >90% purity with high peptide content, and the observed mass for each peptide was in agreement with the calculated mass. The SPPS process for making the analogs is scalable and amenable for optimization, despite the size range of the peptides and modifications introduced in some of the molecules (e.g., N- or C-terminal lipidation).

[0191] The analogs of human NRG-4 are designed and screened based on assays including in vitro receptor dimerization assay (and specificity), metabolic stability assay. In Vitro Assays to Screen for Potency and Specificity

[0192] The analogs, SEQ ID NOs:1-29, were screened for in vitro functional activity using PATHHUNTER® Dimerization assay: DiscoverX ERBB4 / ErbB4 Human RTK Kinase Cell Based Agonist Dimerization Assay (Eurofins DiscoverX Corporation, Catalog # 86-0024P-2715AG), following the manufacturer’s protocols. Human NRG-1 and human NRG-4 were used as positive controls. Human NRG-1 is a potent ErbB4 activator, which has been reported as more potent than NRG- 4 and having a better efficacy response in the PATHHUNTER assay, and therefore is also used as a control by the assay manufacturer.

[0193] Ten-point dose response curves (doses over a 0.3 nM - 6 ^M concentration range, 3 fold dilutions from top dose concentration; n = 2 replicates) of the peptides were generated and the results were averaged to provide EC50values.4878-9453-5582.4Page 37 of 80079065-000004WOPT

[0194] A counter screen to assess receptor specificity was performed using the DiscoverX EGFR / EGFR Human RTK Kinase Cell Based Agonist Dimerization Assay (Catalog# 86-0024P- 2713AG), with a dose concentration range of 3 nM - 50 ^M. This assay utilizes a similar technology to that shown in figure 2, and EGF is used as a positive control. Desirably, NRG-4 analogs are specific in the ErbB family, and should not lead to much EGFR / EGFR dimerization.

[0195] The SAR study was conducted in an iterative manner for optimization of activity and resulted in three rounds of screening, each containing an ErbB4 / ErbB4 assay and an EGFR / EGFR assay. Table 2 presents the full SAR dataset for the ErbB4 / ErbB4 and the EGFR / EGFR Cell Based Agonist Dimerization Assays, with the three screening rounds delineated from each other.

[0196] Findings based on the SAR studies include: (a) SEQ ID NO:1 has truncations at the N- and C-termini while preserving the EGF-like domain. The peptide showed a two-fold improvement (i.e., half the EC50amount) in ErbB4 functional activity compared to parent NRG-4, and had a similar partial agonist profile (compared to efficacy responses induced by reference peptide NRG-1 in ErbB4 receptor homodimerization). Further deletions at the C- terminus of this molecule led to complete loss of functional activity in the ErBB4 / ErbB4 dimerization assay (see SEQ ID NO:12 to SEQ ID NO:14, SEQ ID NO:23). Hence preferably, residue position Xaa50, Xaa49, and Xaa48 are to be retained, not absent. (b) Additional analogs were designed based on SEQ ID NO:1. Lipidation at either the N- or C- terminus provided a set of peptides containing C-14 or C-16 lipid moiety (alkanoyl) or HO2CíRíC(=O)-, wherein R can be C1-C28 aliphatic or aromatic modifications, or R can be C3-C30 aliphatic or aromatic modifications, or R can be C12-C22 aliphatic or aromatic modifications. Some of the analogs included NRG-4 backbone substitutions; and some included a Lys-Ȗ-Glu spacer after the Xaa50residue and before the fatty acid at the C-termini. These analogs (SEQ ID NO:7 to SEQ ID NO:10, SEQ ID NO:16) were either equipotent or were more potent than the parent NRG-4, and displayed similar partial agonist properties as assessed by maximal receptor response. It should be noted that the receptor dimerization assays used BSA as an excipient in the assay medium. Typically, fatty acid- modified peptides bind to this excipient, resulting in right-shifting of binding curves compared to the un-modified peptides (as seen in the GLP-1-based peptide drugs, namely liraglutide (VICTOZA®) and semaglutide (OZEMPIC®)). Thus, the assay results showing equal or higher potencies with N- or C- terminus lipidation compared to SEQ ID NO:1 were unexpected in view of the opposite profile exhibited by lipidated liraglutide and lipidated semaglutide. (c) We probed the effects of changes in the C-terminus end and in the EGF-like domain. Selective combination of modifications at the C-terminus resulted in increased functional activity (SEQ ID NO:15, SEQ ID NO:16) compared to NRG-4 or loss in activity (SEQ ID NO:4 to SEQ ID NO:6, SEQ ID NO:23). Hence, preferably, Xaa50is not changed from Leucine to Proline, and Xaa24is not changed from Tyrosine to Gln (Q) or Val (V); and the tail fragment, Xaa46-Xaa50, of SEQ ID NO:1 is not replaced4878-9453-5582.4Page 38 of 80079065-000004WOPTwith the fragment Asp-Gln-Phe. Also preferably, if fragment Xaa46Xaa47Xaa48Xaa49is Asp46-Gln47- Val48-Phe49(SEQ ID NO:37), then Xaa50is preferably Leucine50, not Proline. The EGF-like domain of NRG-4 is highly sensitive to major changes, such as incorporation of loops from other EGF family members between the disulfide bridges. An example is SEQ ID NO:11, which replaces NRG-4’s fragments between two disulfide loops (a fragment between the first two cysteines, and a fragment between the last two cysteines) with respective fragments from the NRG-3 sequence. This led to a complete loss of functional activity. Hence, preferably, a variant of SEQ ID NO:1 is that fragment Xaa10Xaa11Xaa12Xaa13Xaa14Xaa15Xaa16is not Arg-Asp-Lys-Asp-Leu-Ala-Tyr (SEQ ID NO:38), and that Xaa37Xaa38Xaa39Xaa40Xaa41Xaa42Xaa43Xaa44is not Lys-Glu-Gly-Tyr-Gln- Gly-Val-Arg (SEQ ID NO:39). However, selective amino acid substitutions in the EGF-like domain are tolerated and the functional activity is further modulated when combined with C-terminal changes (SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:22). Hence, preferably, a variant of SEQ ID NO:1 is that Xaa19of SEQ ID NO:1 is changed from Asparagine19(N) to Histidine (H); that Xaa46Xaa47of SEQ ID NO:1 is changed from Glu46-Glu46to Asp-Gln; that Xaa20of SEQ ID NO:1 is changed from Glycine20(G) to Aspartate (D); and / or Xaa46Xaa47Xaa48Xaa49Xaa50of SEQ ID NO:1 is replaced with Asp-Gln-Phe-Leu-Leu (SEQ ID NO:40). The combination of such changes in the backbone translated to an increase in potency and efficacy (maximal receptor response). Thus, SEQ ID NO:17 has two times the potency compared to native NRG-4 (EC50 of 19.2 nM vs ~40 nM) and shows a superior efficacy response (90% vs 44-55% maximal receptor response). (d) THE SAR study shows that NRG-4’s interaction with ErbB4 is finely tuned. Incorporation of a D-enantiomeric amino acid at the C-terminus, as shown in SEQ ID NO:27, to prevent chymotrypsin degradation resulted in a complete loss of receptor potency. Note, comparing SEQ ID NO:17 and SEQ ID NO:27, wherein the only difference is in Xaa49 which changes from L-enantiomeric phenylalanine (F) to D-enantiomeric phenylalanine (d-F). This highlights that the interaction of ErbB4 with those amino acids flanking the EGF-like domain is exquisitely sensitive to chirality, and the termini play an important role for eliciting functional activity as well. Hence, preferably, Xaa46, Xaa47, Xaa48, Xaa49, and Xaa50are all L-enantiomeric amino acids, not D-enantiomeric amino acids. Further alterations of EGF-like domain and C-terminal regions did not improve upon potency as illustrated by SEQ ID NO:24 (as opposed to SEQ ID NO:21), SEQ ID NO:25 (as opposed to SEQ ID NO:21), and SEQ ID NO:26 (as opposed to SEQ ID NO:17 and SEQ ID NO:16). Hence, preferably, a variant of SEQ ID NO:1 is that when Xaa19of SEQ ID NO:1 is replaced with Histidine (H) or when fragment Xaa19Xaa20of SEQ ID NO:1 is replaced with His-Asp, then Xaa47is not replaced with Gln (Q). Also, when a variant of SEQ ID NO:1 contains both a substituted fragment of Xaa46Xaa47Xaa48Xaa49Xaa50, Asp46-Gln47-Val48-Phe49-Leu50(SEQ ID NO:41), and a substituted Xaa19,4878-9453-5582.4Page 39 of 80079065-000004WOPThistidine19(H), then this variant does not have lipidation on the N terminus (not lipidated on the N terminus). (e) Analogs such as SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:22 retain the high selectivity for ErbB4 receptor over EGF receptor, as compared to that exhibited by SEQ ID NO:1. That is, SEQ ID NO:1 has about 1000-fold difference between the EC50for ErbB4 / ErbB4 dimerization and the EC50for EGFR / EGFR dimerization, whereas this difference is much greater (more than 1,000-fold and up to about 2,680-fold) seen in SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:22.

[0197] Nine NRG-4 analogs, along with NRG-4(5-50) were screened in an ERBB2 / ERBB4 Human RTK Kinase Cell based Agonist Hetero-Dimerization Assay (Catalog # 86-0024P-2714AG). Table 3 presents the results from this ERBB2 / ERBB4 Hetero-Dimerization Assay. Heregulin-B1 was used as a positive control, as it promotes heterodimerization of ErbB2 and ErbB4 subunits and is a full agonist with an EC50of 0.15 nM. The “parent” NRG-4 (NRG-4(1-62)) is a weaker ligand and partial agonist, inducing receptor heterodimerization with an EC50of 5.7 nM, which is in line with literature findings. Like the parent molecule, the selected NRG-4 analogs induced receptor heterodimerization as well, with EC50values ranging from 1.4 - 8.2 nM, and in general exhibited as partial agonists in the assay. Overall the analogs were very similar to NRG-4(1-62). Table 1. Synthesized analogs of NRG-4 and their characterization.4878-9453-5582.4Page 40 of 80079065-000004WOPT4878-9453-5582.4Page 41 of 80079065-000004WOPTTable 2. ERBB4 / ERBB4 & EGFR / EGFR ASSAYS.4878-9453-5582.4Page 42 of 80079065-000004WOPT* Response refers to the chemiluminescent signal from the functional enzyme reaction upon dimerization of receptor subunits. Table 3. ERBB2 / ERBB4 ASSAY FOR NRG-4 ANALOGS SUBSET4878-9453-5582.4Page 43 of 80079065-000004WOPTsn.o)iea ^1 ^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^S^StilsbaoaX 3tea ^ haX03^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^P^Pe dmasirceeA^a ^ tifoa 0 ^G^G^G^G^G^G^G^G^G^G^G^R^G^G^G^G^G^G^G^G^G^G^ ^ ^ ^i1 G G G Gntni Xosn m a^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^eecdA X9a C C C C C C C C C C C C C C C C C C C C C C C C C Ci ^ n^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^e OaX8a P P P P P P P P P P P P P P P P P P P P P P P^P^P^PuqN a^7 ^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^E^ ^ ^ ^eSDI X a E E E E. ^ sQ a 6 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^eE X a E E E E E E E E E E E E E E E E E E E E^E^E^E^E^E^Ecn S^ e. a 5 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^uB H H H H H H H H H H H H H H H H H^H^H^H^H^H^H^H^ ^1X a H Hqe- ^ s4 aX4 ^D^^^^^^^^^^^^^D^D^^^^^^^^^^^^^^^D^^^^^^^^^^^^^^^^^^eeadl^ i^tb a 3T^^^^^^^^^^^^^G^G^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^paeTX a Gpynli^^^ 2d ^^^^ a^P^^^^^^^^^^^6^1iCc4da1ic ^^^^^^^6d^^^^^^^1ic ^^^^^^^^^^^ ^ ^ ^ona iC aiC ai^ ^ ^ ^PX d d d.woAh a^1^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^ ^ ^1s X aM^^ ^ ^ ^-er^4 aaea^ l2X)(0^^ ^ ^ ^ ^ ^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ b6a-^^ ^ ^ ^ ^ ^^^^^^^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^^ ^ ^^^ ^ ^ ^ ^^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^^^^^T23ES QDI N:O^^03 1 2 3 4 5^6^7^8^901112131415161718191021222324252FF F F F L L s dpahn eh.^ ^ ^uV VteV^V^ ^V^ ^^^ ^^^^F^ ^^F 41 ofdi :pot^ Q ^^E^ ^^E^ ^ ^ ^ ^ ^ OmQ ^ ^ Q ^ Q ^ ^ QNos pcu epa ni y^D loD^^E^ ^^E^ ^^D ^ ^^D ^^D ^ ^^D Io mQtre pt-eh^C^^C^ ^^C^ ^^C^ ^^ E srC^^C^ ^^C S. eNtfs fer eh ots^ nR ^^R^ ^^R^ ^^R^ ^^R^^R^ ^^R os)il tur y aniaodm^A^^A^ ^^A^ ^^A^ ^^A^^A^ ^^A p nma ecn roet-^ ^oeiG Gcdta is CG^G^ ^^G ^ ^^G ^ ^^G ^^^ ^^ ec toe^n peT^^T^ ^^T^ ^^T^ ^^T^^T^ ^^T eu hphqy st itaex^o ytdeY ^^Y^ ^^Y^ ^^Y^ ^^Y^^Y^ ^^sYbeni riao noc miti^y- N ^^N ^ ^^N ^ ^^N ^ ^^N ^^N ^ ^^Ngo 7 d sol 1-npuuos^E^^E^ ^^E^ ^^E^ ^^E^^E^ ^^o iEml poGyh-Ȗmt- oe^V^V^V^r c cio^ ^ ^ ^ ^V^ ^^V^^V^ ^^Vof Asim pE h^C u E t dd nnC ^^C^ ^^C^ ^^C^ ^^C^^C^ ^^eAun- eRR^R^ ^R^ ^R^R^ ^Ric howp^ ^^^^ ^ ^ ^ ^l A m erE tEnoec^ ^^^ ^^^ ^^^ ^^^^^^ as AnosiC C C C C C^C e (ht^F^^F^ ^^F^ ^^ n sy pF^ ^^F^^F^ ^^F ietmnsL,o e^P^^P^ ^^P^ ^^P^ ^^P^^P^ ^^Pycyc hlelewd l^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^hatni:otyitpe kla^92^ ^84^ ^ ^94^ ^ ^05^ ^ ^15^ ^25^ ^ ^3e5tiodpdyl6- N Aop1C(Formula II) Metabolic Stability in Simulated Gastric and Intestinal Fluids

[0198] Metabolic stability studies of NRG-4 analogs were performed for those exhibiting promising functional receptor activity profiles demonstrated above. Stability in simulated GI fluids was assessed by peptide incubation in buffers mimicking gastric and intestinal pH in the presence of the GI enzymes, chymotrypsin, trypsin and pepsin. Incubated peptide samples were analyzed for intact peptide by LC-MS at various time points. Desmopressin and somatostatin were used as control peptides for the studies, and parent NRG-4 (NRG-4(1-62)) will be used as a comparator. Salmon calcitonin could alternatively or also be used as a control peptide. Simulated intestinal fluid (SIF) was prepared by adding a metabolic enzyme, such as chymotrypsin or trypsin, to phosphate buffer pH 6.8. Simulated gastric fluid (SGF) was prepared by addition of pepsin to NaCl solution adjusted to pH 1.2 with HCl and pre- heated to 37 °C.

[0199] Specifically, the SIF studies were performed in duplicate, using chymotrypsin as a metabolic enzyme, for SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:22. These polypeptides (as NRG-4 analogs), and NRG-4, and control peptides were individually added to SIF at 37 °C. Aliquots were removed at time points between t = 0 and t = 240 min, and he enzyme activity was quenched by the addition of trifluoroacetic acid (TFA).

[0200] Figure 3 shows that whereas somatostatin is rapidly cleaved by chymotrypsin, desmopressin has a slower rate of degradation (dotted curves). NRG-4 displays an intermediate degradation profile. SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:15 are similar to the parent peptide, NRG-4, in terms of stability, whereas SEQ ID NO:17 and SEQ ID NO:21 undergo slightly faster cleavage. Figure 4 shows that SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:16 and SEQ ID NO:22 are 4878-9453-5582.4 Page 48 of 80079065-000004WOPTmore resistant to chymotrypsin compared to the parent NRG-4. Therefore, NRG-4 analogs that are functionally more active at the ErbB4 receptor than NRG-4 and also more stable in the presence of enzymatic degradation have been identified.

[0201] These analogs were also studied for stability in SIF containing trypsin and in simulated gastric fluid (SGF) containing pepsin and were found to be more stable than parent NRG-4 in SIF containing trypsin (FIG.5) and in SGF containing pepsin (FIG.6A).

[0202] SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:22 have been identified for having (a) a significantly smaller in size than human NRG-4, (b) ~two-fold more potent (or at least about two times the potency) in functional activity at the ErbB4 receptor compared to NRG-4, and display a higher maximal receptor response, (c) specific ligands for ErbB4, with greater than 1000-fold selectivity for the receptor compared to EGFR, and (d) several of these analogs show superior metabolic stability compared to parent NRG-4 in the SIF-chymotrypsin assay, while most are superior in metabolic stability in SIF-trypsin and SGF-pepsin assays.

[0203] Metabolic Stability of Selected NRG-4 Analogs in Simulated Gastric Fluids (SGF): Metabolic stability studies of NRG-4 analogs, selected for their promising functional receptor activity profiles, were performed using SGF.

[0204] The studies were conducted according to the following general procedure. Stability in SGF was assessed by peptide incubation in a buffer mimicking gastric pH in the presence of pepsin. Incubated peptide samples were analyzed for intact peptide by LC-MS at various timepoints. Desmopressin and somatostatin were used as control peptides for the studies, and parent NRG-4 was used as a comparator. Salmon calcitonin as a control peptide is not included as originally proposed, as it is currently not available from vendors. SGF is prepared by addition of pepsin to NaCl solution adjusted to pH 1.2 with HCl and pre-heated to 37qC.

[0205] Figure 6A shows that whereas NRG-4 is rapidly cleaved by pepsin, the NRG-4 analogs are significantly more stable compared to the parent peptide under the same conditions.

[0206] Mouse and Human Plasma Stability Studies. The six analogs were also screened for metabolic stability in human and mouse plasma. The peptide analogs were incubated in these biological matrices at 37qC, and samples were withdrawn at 15, 60, 120 and 180 min and analyzed by RP-HPLC.

[0207] Figures 7A and 7B show that all the analogs were highly stable in mouse and human plasma.

[0208] Physicochemical Characterization of Analogs: Peptide solubility studies were conducted to meet the solubility and stability requirements for in vivo studies. The analogs were assessed at 3 different physical and chemical stability at 4°C of at least two weeks and at RT for >3 days to support dose preparation.

[0209] Studies indicated that 40 mM Tris Buffer, 5 mg / mL mannitol at pH=7.5 was suitable for the selected NRG-4 analogs. Solubility was up to 2.5 mg / mL of each peptide prior to dilution to 0.54878-9453-5582.4Page 49 of 80079065-000004WOPTmg / mL. SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:22 were soluble at 0.5 mg / mL in 40 mM Tris buffer, 5 mg / mL mannitol at pH=7.5 and remained clear and colorless after three freeze and thaw cycles.

[0210] The peptide formulations were incubated for 2 weeks in the freezer at -20 °C and in the refrigerator at 2-8 °C and peptide purity was determined by HPLC after 2 weeks of storage. There was no degradation observed when the liquid formulations were stored at -20 °C for at least 2 weeks. The same results were found for all six peptides after 2 weeks of storage in the refrigerator at 2-8 °C except for SEQ ID NO:8 and SEQ ID NO:21 which degraded by about 3% and 2%, respectively, in two weeks (Figure 8). Example 2 In Vivo Screening Using a Mouse DSS Model Screening to Identify Efficacious NRG-4 Analogs.

[0211] Analogs from Example 1 (e.g., SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:22) are tested for efficacy in the dextran sulfate sodium (DSS)-induced colitis mouse model (e.g., male C57Bl / 6 mice) using intra-cecal administration (to mimic intestinal drug release) to deliver controlled drug load to the large intestine. Analogs will then be tested in dose response studies, with parent NRG-4 at a comparator dose. Efficacy is assessed by histopathological analysis (primary endpoint), with video endoscopy and disease activity index scores (secondary endpoints). In addition, the efficacy of the selected lead NRG-4 analog is independently confirmed in a T cell-mediated chronic colitis model. Table 5-1. Efficacy study design using the DSS mouse model. IC: Intra-Cecal.1Sham animals will undergo surgery and antibiotic treatment but with no cannula placement.2Per mouse.3Test article will be administered in 0.075 mL / animal IC. Table 5-2. Disease Activity Index scoring scheme. 4878-9453-5582.4Page 50 of 80079065-000004WOPT

[0212] The intra-cecal delivery approach bypasses the stomach and achieves controlled drug loads via the cecal cannula to the colon region. At least 10 days prior to the start of the experiment, a sufficient cohort of mice (83 animals) will undergo surgical implantation of a cecal cannula to satisfy the requisite number (75 mice) to be enrolled and randomized in the main study. Table 5-1 shows the general efficacy study design for screening of NRG-4 analogs. Prior to analog screening, parent NRG- 4 will be tested at 3 doses (200, 500 and 1500 μg / kg) to pick a comparator dose for analog screening. Colitis will be induced in C57Bl / 6 mice by exposure to 3% DSS-treated drinking water from Day 0 to Day 5. One additional group of mice will serve as a no-disease control (Group 1). Mice will be dosed with vehicle (Group 2), NRG-4 (Group 3) or NRG-4 analogs (Groups 4-6) via intra-cecal administration (IC) at a volume of 0.075 mL / animal QD from Day 6 to Day 19. All animals will be weighed daily and assessed visually for the presence of diarrhea and / or bloody stool at the time of dosing. The severity of weight loss, diarrhea, and blood in stool will be assessed on Days 0, 6, 12, and 21. Each of these parameters are individually scored (examiner blinded to treatment groups) on the above days according to the disease activity index (DAI) scoring scheme shown in Table 5-2, yielding DAI scores and serving as a secondary endpoint, with a minimum of 0 (>4.99% body weight loss, no diarrhea, no blood in stool) and a maximum of 12 (>20% body weight loss, diarrhea, and gross bleeding). Each mouse will undergo video endoscopy on Days 12 and 19 to assess the severity of colitis using a small animal endoscope (Karl Storz Endoskope, Germany) while under isoflurane anesthesia. During each endoscopic procedure, still images as well as video will be recorded to evaluate the extent of colitis and the response to treatment. Additionally, we will attempt to capture an image from each animal at the most severe region of disease identified during endoscopy. This additional measure of colitis severity will be scored (examiner blinded to treatment groups) using a 0-4 scale (0= normal; 1= loss of vascularity; 2= loss of vascularity and friability; 3= friability and erosions; 4= ulcerations and bleeding). As an additional secondary endpoint, stool consistency will be scored during the endoscopy also using a 0-4 scale (0= normal, well-formed pellet; 1= loose stool, soft, staying in shape; 2= loose stool, abnormal form with excess moisture; 3= watery or diarrhea; 4= bloody diarrhea). Feces will be collected and frozen on Day 19 to allow for possible subsequent assessment of lipocalin-2 as an inflammation biomarker. Following4878-9453-5582.4Page 51 of 80079065-000004WOPTendoscopy on Day 19, animals from all groups will be euthanized by CO2 inhalation and will undergo terminal blood and colon collection. Plasma will be collected from the terminal blood sample (centrifugation at 4,000 × g for 10 minutes) using K2EDTA as the anti-coagulant, flash frozen, and stored at -80°C. The colon will be rinsed in PBS, measured, weighed, and then trimmed to 5 cm in length and divided into 3 pieces. The most distal and proximal 2 cm sections will each be placed in 10% neutral buffered formalin for fixation for subsequent histological evaluation. The middle 1 cm portion will be placed into a cryotube, weighed, and snap frozen in liquid nitrogen. The plasma and frozen colon tissue will be stored at -80°C until needed for additional endpoint analysis. Paraffin block processing, sectioning and H&E staining will be performed . A tissue slide of each colon will be scored by a board-certified pathologist (who will be blinded to the group designations) at HistoTox Labs using histological scoring criteria described in Progress Report Section 6. Histopathology scoring will serve as the primary endpoint for the studies. Data will be analyzed by either one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to Group 2 (vehicle control) or by the Kruskal-Wallis test with Dunn’s multiple comparisons test to compare all groups to Group 2 (vehicle control). All statistical analyses will be performed by a biostatistician at HistoTox Labs using GraphPad Prism 8 (La Jolla, CA). The group size of n = 15 in these studies will provide 90% power to detect at least a 25% reduction of the variables (ANOVA, set at 0.05). A lead NRG-4 candidate will be chosen based on the best combination of in vitro and in vivo efficacy, metabolic stability and physicochemical properties. Example 3 Selection of Analogs for In Vivo Studies

[0213] The selection of analogs for in vivo efficacy studies were based on the criteria of superior functional activity and enhanced metabolic stability compared to native NRG-4, and favorable physicochemical characteristics as determined from the formulation studies. A summary of the selection rationale is shown in Table 6. Table 6. Analog selection criteria for in vivo studies.4878-9453-5582.4Page 52 of 80079065-000004WOPT

[0214] A key consideration was that, with the other factors being comparable, the analogs had to show the good GI metabolic stability for oral delivery considerations. SEQ ID NO:8, SEQ ID NO:22 and SEQ ID NO:16 (SEQ ID NO:8 and SEQ ID NO:16 are lipidated) displayed the highest metabolic stability across the assays. SEQ ID NO:15 was more stable than NRG-4 in trypsin and pepsin assays and was comparable to NRG-4 in its stability to chymotrypsin. While SEQ ID NO:17 was more stable than NRG-4 in trypsin and pepsin assays, it is rapidly degraded by chymotrypsin. NRG-4 analogs SEQ ID NO:22, SEQ ID NO:15 and SEQ ID NO:16 were selected for further studies.

[0215] SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17 were synthesized in 100 mg scale at CPC Scientific as their acetate salts, with their peptide purity (as determined by RP- HPLC) and peptide content (as determined by amino acid analysis) exceeding 90% & and 85% respectively. All the peptides were further characterized by LC / MS and displayed the correct molecular weights. Example 4

[0216] Efficacy Screening Using a Mouse DSS Model. As described in Example 2, the studies were performed and the results are described herein.

[0217] The objective of these studies was to evaluate the efficacy of the selected NRG-4 analogs in a DSS-induced colitis model. The screening studies were conducted at a CRO that has specifically developed an intra-cecal administration route using male C57Bl / 6 mice in this widely used IBD model for initial assessment of efficacy of colon-directed peptidic candidates. The intra-cecal delivery approach bypasses the stomach and achieves controlled drug loads via the cecal cannula to the colon region. Efficacy was assessed by pathological analysis (primary endpoint), and video endoscopy and disease activity index scores (secondary endpoints). Prior to analog screening, parent NRG-4 was tested at 3 doses (200, 500 and 1500 ^g / kg) to pick a comparator dose for analog screening. The details of the study design are shown in Table 7. Table 7: Study Design.1Sham animals underwent surgery and antibiotic treatment, but had no cannula placement.2Per mouse. Test Article was administered in 0.075 mL / 20g animal IC.4878-9453-5582.4Page 53 of 80079065-000004WOPT

[0218] Study Design: A minimum of 10 days prior to the start of the experiment the animals in Groups 2-5 underwent surgical implantation of a cecal cannula. A sufficient number of animals underwent implantation to allow for a total of 60 cannulated animals (plus 5 sham animals in Group 1) to be enrolled in Groups 2-5 of the study. Colitis was induced in 60 male C57Bl / 6 mice by exposure to 3% DSS-treated drinking water from Days 0 to 5. Animals in Group 1 served as sham, no disease controls. Group 2 was dosed IC with vehicle QD from Days 6-19. The animals in Groups 3-5 were treated with NRG-4 at 200, 600, or 1500 ^g / kg via IC from Days 6-19, respectively. All animals were weighed daily and assessed visually for the presence of diarrhea and / or bloody stool at the time of dosing. Each of these parameters was individually scored according to the disease activity index (DAI) scoring scheme shown in Table 8, yielding DAI scores with a minimum of 0 (^.99% body weight loss, no diarrhea, no blood in stool) and a maximum of 13 (>30% body weight loss, diarrhea, and gross bleeding). Any animal that lost >30% of its body weight, showed an inability to eat, or was moribund, was be euthanized without sample collection. Mice underwent video endoscopy on Days 12 and 19 to assess colitis severity using a small animal endoscope (Karl Storz Endoskope, Germany) while under isoflurane anesthesia. During each endoscopic procedure, still images as well as video were recorded to evaluate the extent of colitis and the response to treatment. Additionally, images were captured from each animal at the most severe region of disease identified during endoscopy. This additional measure of colitis severity was scored (examiner blinded to treatment groups) using a 0-4 scale at the most severe region observed (0= normal; 1= loss of vascularity; 2= loss of vascularity and friability; 3= friability and erosions; 4= ulcerations and bleeding). Additionally, stool consistency was scored during endoscopy using the parameters defined in Table 9. Table 8Table 94878-9453-5582.4Page 54 of 80079065-000004WOPT

[0219] Following endoscopy on Day 19, animals from all groups were sacrificed for terminal sample collection. Terminal blood was collected via cardiac puncture and prepared for plasma. The colon of each animal was excised, measured, rinsed, and weighed. The most distal 5 cm portion was isolated. The most distal and proximal 1.5 cm pieces were fixed in 10% NBF for possible histopathology. The middle 2 cm section of colon was weighed, snap frozen, and stored at -80°C for potential downstream analyses.

[0220] Statistical Analyses. Data was analyzed by either one-way ANOVA with Dunnett’s multiple comparisons test used to compare all groups to Group 2 (vehicle) or Kruskal-Wallis test with Dunnett’s multiple comparisons test used to compare all groups to Group 2 (vehicle). All statistical analyses were performed using GraphPad Prism 9.3.1 (La Jolla, CA).

[0221] Results & Conclusions: Animals in which colitis was induced by exposure to 3% DSS demonstrated mean body weight loss from Days 5 to 8 (Fig.9). Body weight increased thereafter, returning to baseline by study end. Overall body weight gain was higher in naïve animals (Group 1) as compared to diseased mice treated with vehicle (Group 2), though the difference was not statistically significant. Among diseased groups, overall body weight gain was elevated in animals treated with 200 μg / kg NRG-4 (Group 3) and 1500 μg / kg NRG-4 (Group 5) as compared to animals treated with vehicle. In order to determine the statistical significance of overall differences in mean percent body weight change between groups, the area under the curve (AUC) was calculated using the trapezoidal rule transformation. The differences were not statistically significant for the treatment groups when compared to vehicle control. Similarly, individual-day analyses did not reveal statistical differences.

[0222] There were no major differences on overall DAI scores when comparing between diseased groups (data not shown). Animals underwent endoscopy on Days 12 and 19 in order to assess colonic inflammation. Animals in which colitis was induced demonstrated increased mean colitis severity and stool consistency scores relative to naïve animals on both Days 12 and 19. Both colitis severity and stool consistency scores were significantly lower in naïve animals as compared to the vehicle control group at both timepoints. Mean colitis severity score was notably decreased by 15% in animals treated with 200 μg / kg and 1500 μg / kg NRG-4 as compared to animals treated with vehicle, though neither decrease reached statistical significance (Figure 10). Decreased stool consistency scores were observed in these same groups on Day 12. There were no major differences in mean colitis severity score or stool consistency score between diseased groups on Day 19.

[0223] No major differences in mean colon weight:length ratio were observed when comparing groups in which disease was induced. Based on the results from the NRG-4 dose response4878-9453-5582.4Page 55 of 80079065-000004WOPTstudy using IC delivery, 1500 μg / kg NRG-4 dose was selected as a comparator dose for the analog screening studies.

[0224] Studies to Evaluate the Efficacy of NRG-4 Analogs in DSS-Induced Model of Colitis in C57BL / 6 Male Mice: SEQ ID NO:22, SEQ ID NO:15 and SEQ ID NO:16 at 3 doses (200, 500 and 1500 ^g / kg) were tested in the DSS induced mouse colitis model using μg / kg NRG-4 dose as a comparator dose. Table 10 shows the study design. The experimental protocol and scoring criteria were identical to that described earlier, and the results for SEQ ID NO:22 are discussed below. Table 10: Study Design1Sham animals underwent surgery and antibiotic treatment but had no cannula placement.2Each Analog was run in an independent study.3Per mouse. Test Article was administered in 0.075 mL / 20 g.

[0225] Results. SEQ ID NO:15 displayed modest efficacy and was comparable to parent NRG-4. SEQ ID NO:16 showed some efficacy in this animal model. It is possible that its lipid modification sequesters the peptide more than needed to reduce its availability in the GI tract. Overall, SEQ ID NO:22 displayed superior efficacy when compared to a high dose of parent NRG-4. SEQ ID NO:22 is referred to as NRG-4 Analog 1 in the graphs. Data were evaluated using either one-way or two-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle control group. All statistical analyses were performed using GraphPad Prism 9.4.1 (La Jolla, CA). Statistical significance was achieved when p<0.05.

[0226] SEQ ID NO:22 Studies- Body Weight. Animals were weighed daily for the duration of the study and the percent body weight change relative to Day 0 (Figure 11, upper) was calculated. As before, the area under the curve (AUC) was calculated using the trapezoidal rule transformation to determine the statistical significance of overall differences in mean percent body weight change4878-9453-5582.4Page 56 of 80079065-000004WOPTbetween groups. This is shown over the entire study period in the Figure 11 (right-hand side). An additional day-by -day analysis was performed for Days 6-12 and is shown in Table 11.

[0227] Diseased animals (Groups 2-6) demonstrated notable weight loss between Days 5 and 9, followed by weight recovery (Figure 11). Disease-naive sham animals (Group 1) did not demonstrate this weight loss and overall displayed significantly increased weight gain (p<0.01) relative to diseased and cannulated animals treated with vehicle (Group 2). Treatment with 600 μg / kg SEQ ID NO:22 (Group 5) resulted in notable, though non-statistically significant, improvements to weight loss relative to treatment with vehicle alone.

[0228] Additionally, transient weight loss was observed on Day 13 in all groups, likely due to endoscopy under anesthesia on Day 12 and associated stresses and handling.

[0229] Weight change was also evaluated from Days 5-19, omitting Days 0-4 during disease induction. Similar trends were present; however, treatment with 200 μg / kg SEQ ID NO:22 (Group 4) produced the greatest beneficial effect on weight loss relative to treatment with vehicle alone (Figure 11, lower). Table 11. Individual Day analysis of Percent Body Weight Change

[0230] Percent weight change from Day 0 baseline was also assessed day-by-day from Days 6-12 by comparing all groups to the vehicle control group (Group 2); analysis is shown in Table 12. Disease-naïve sham animals (Group 1) displayed significantly increased weight change on Days 6-11, compared to animals treated with vehicle (Group 2). Animals treated with 1500 μg / kg NRG-4 parent demonstrated significantly increased weight gain on Day 9 when compared to animals treated with vehicle. Animals treated with 200 (Group 4) and 1500 (Group 6) μg / kg SEQ ID NO:22 demonstrated significantly increased weight gain on Days 8 and 9 when compared to animals treated with vehicle (Group 2). Animals treated with 600 μg / kg SEQ ID NO:22(Group 5) demonstrated significantly increased weight gain on Days 6, 7, 8, 9, and 10 when compared to animals treated with vehicle.4878-9453-5582.4Page 57 of 80079065-000004WOPT

[0231] SEQ ID NO:22 Studies- Body Weight Disease Activity Index. Animals were observed daily for weight loss, diarrhea, and blood in stool. Composite DAI scores over time are shown in Figure 9. In order to determine the statistical significance of overall differences in mean composite DAI score between groups, the area under the curve (AUC) was calculated over the entire study period using the trapezoidal rule transformation and is shown in the inset. An additional day-by -day analysis was performed for Days 6-12 and is shown in Table 12. Diseased animals demonstrated elevated mean composite DAI scores, particularly between Days 6 and 13. Naïve animals, none of which displayed composite DAI scores above 0 at any time, displayed significantly lower composite DAI scores when compared to animals treated with vehicle by AUC analysis (p<0.05). Animals treated with test articles (Groups 3-6), particularly those treated with 600 and 1500 μg / kg SEQ ID NO:22, demonstrated decreased composite DAI scores relative to animals treated with vehicle alone, though these comparisons were not found to be statistically significant.

[0232] Composite DAI scores were also assessed day-by-day from Days 6-12 by comparing all groups to the vehicle control group (Group 2); analysis and p values are shown in Table 7. Disease- naïve sham animals (Group 1) displayed significantly lower composite DAI scores on Days 6, 8, 9, and 12, compared to animals treated with vehicle (Group 2). Animals treated with 200 μg / kg SEQ ID NO:22 (Group 4) demonstrated significantly decreased composite DAI scores on Day 8; animals treated with 600 μg / kg SEQ ID NO:22 demonstrated significantly decreased composite DAI scores on Days 6, 8, 9, and 12; and animals treated with 1500 μg / kg SEQ ID NO:22 demonstrated significantly decreased composite DAI scores on Days 6 and 8. Table 12. Individual Day Analysis of DAI Scoring.

[0233] SEQ ID NO:22 Studies: Endoscopy and Stool Consistency Scores. Animals underwent endoscopy on Days 12 and 19 in order to assess colonic inflammation. Stool consistency was likewise scored visually on a five-point scale as described in Table 9. On both Day 12 and Day 19 endoscopy (Figure 13 and Figure 14, respectively), naïve animals displayed lower colitis severity and stool consistency scores, relative to diseased animals treated with vehicle. Naïve animals displayed4878-9453-5582.4Page 58 of 80079065-000004WOPTsignificantly lower colitis severity scores on Day 12 (p<0.01), and on Day 19 they displayed notably lower colitis severity scores, though this comparison to animals treated with vehicle did not reach statistical significance (p=0.0669). Naïve animals also displayed significantly decreased stool consistency scores on Day 12 relative to animals treated with vehicle (p<0.01). Animals treated with 200 μg / kg SEQ ID NO:22 (Group 4) demonstrated a strong reduction (30%), and nearly statistically significant (p=0.0505), reduction in the mean colitis score on Day 12 endoscopy when compared to animals treated with vehicle.

[0234] Animals treated with 1500 μg / kg NRG-4 parent demonstrated significantly reduced colitis scores (39%) on Day 19 endoscopy when compared to animals treated with vehicle (p<0.01). Also of note, and clinically relevant, a 24% reduction in the mean colitis score was observed after treatment with 600 μg / kg SEQ ID NO:22 on Day 19 compared to the vehicle control group. An 18% reduction in the mean colitis score was also observed with the other SEQ ID NO:22 doses levels, 200 and 1500 μg / kg, compared to the vehicle control group.

[0235] SEQ ID NO:22 Studies: Colon Measurements at Euthanasia. All surviving animals were euthanized on Day 19. The colon was excised, measured, rinsed, and weighed, and the colon weight:length ratio was calculated (Figure 15A). Animals in which colitis was induced demonstrated increased mean colon weight:length ratios as compared to naïve animals. A significant increase in the mean colon weight:length ratio was observed in the diseased animals that were treated with vehicle, as compared to the naïve, sham control group. The animals that were treated with 600 μg / kg SEQ ID NO:22 demonstrated a significantly decreased colon weight:length ratio relative to diseased animals treated with vehicle (p<0.05).

[0236] Conclusion: Treatment with 600 μg / kg SEQ ID NO:22 and 200 μg / kg SEQ ID NO:22 resulted in notable, though non-statistically significant, improvements to weight loss relative to treatment with vehicle alone. Treatment with 200 (Days 8 & 9), 600 (Days 6-10), and 1500 (Days 8 & 9) μg / kg SEQ ID NO:22, along with treatment with 1500 μg / kg NRG-4 parent (Day 9), resulted in significant increases in weight gain on the specified days, relative to treatment with vehicle alone.

[0237] Animals treated with test peptides, particularly those treated with 600 and 1500 μg / kg SEQ ID NO:22, demonstrated decreased composite DAI scores relative to animals treated with vehicle alone, though these comparisons were not found to be statistically significant. Treatment with 200 (Day 8), 600 (Days 6, 8, 9, and 12), and 1500 (Days 6 & 8) μg / kg SEQ ID NO:22 resulted in significant decreases in composite DAI scores on the specified days relative to treatment with vehicle alone.

[0238] Animals treated with 200 μg / kg SEQ ID NO:22 demonstrated notably (although non- significantly; 30%) reduced colitis scores on Day 12 endoscopy when compared to animals treated with vehicle. Animals treated with 1500 μg / kg NRG-4 parent demonstrated significantly reduced colitis scores on Day 19 endoscopy when compared to animals treated with vehicle. Additionally, reductions4878-9453-5582.4Page 59 of 80079065-000004WOPTof 18-24% in mean colitis severity scores were observed after treatment with SEQ ID NO:22 at all dose levels on Day 19 compared to treatment with vehicle.

[0239] Animals treated with 600 μg / kg SEQ ID NO:22 demonstrated significantly decreased colon weight:length ratios relative to diseased animals treated with vehicle. These studies therefore demonstrate that SEQ ID NO:22 is superior to parent NRG-4. The results indicate that SEQ ID NO:22 has (1) greater efficacy and potency at the ErBB4 receptor while retaining its receptor specificity, (2) improved metabolic stability in simulated gastric and intestinal fluids compared to the parent peptide and high stability in mouse and human plasma, (3) smaller size (46 vs 62 residues, significant ease of synthesis using solid phase peptide synthesis), and (4) improved efficacy in the DSS-induced mouse model of colitis using an intracecal route of administration. Example 5 Efficacy Using a Mouse T cell-mediated Chronic Colitis Model (Naïve THCell Transfer Mouse Colitis Model).

[0240] We used the adoptive T cell transfer colitis model. While inflammation is driven by adoptive transfer of naïve T cells, it is associated with massive monocyte-derived inflammatory macrophage recruitment to the gut. Literature review reveals a probable initial role of macrophages in the pathogenesis of this chronic colitis model. We conceive that NRG-4 analogs would be effective in this model based on its suppressive action on M1 macrophages and inhibition of TNFĮ and INFȖ production.

[0241] The polypeptide having an amino acid sequence of SEQ ID NO:22 was selected for this study in the adoptive T cell transfer mouse colitis model, based on its superior combination of in vitro and in vivo efficacy, metabolic stability, and physicochemical properties, shown above. The intra- cecal delivery approach bypasses the stomach and achieves controlled drug loads via the cecal cannula to the colon region. Herein, the efficacy was evaluated of different doses of the polypeptide whose amino acid sequence is shown in SEQ ID NO:22 delivered by cecal cannula in the treatment of colitis induced through adoptive transfer of a subpopulation of CD44- / CD62L+ T Cells isolated from C57Bl / 6 donor mice into RAG2- / - recipients. Parent NRG-4 was used as a comparator at 1500 μg / kg dose.

[0242] Animals: Male RAG2- / - recipient mice (n=80; 6-8 weeks) with an average starting body weight (± SEM) of 23.9±1.5 g were obtained from Taconic Biosciences (Rensselaer, NY). Donor male C57Bl / 6 mice (n=57; 6- 8 weeks) were obtained from Taconic Biosciences (Rensselaer, NY). Animals were acclimatized prior to study commencement. During this period, the animals were observed daily in order to reject any that presented in poor condition.

[0243] Housing: The study was performed in animal rooms provided with HEPA filtered air at a temperature of 70±5 °F and 50%±20% relative humidity. Animals were housed in groups of 5-15 per cage. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light / dark cycle of 12 hours on and 12 hours off with no twilight.4878-9453-5582.4Page 60 of 80079065-000004WOPTALPHA-DRI® or equivalent bedding was used; bedding was changed every two (2) weeks, with ¼ of dirty cage material captured and transferred to the new cage. Cages, tops, and water bottles were washed with a commercial detergent and allowed to air dry. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number, and treatment group was used to mark all cages. The temperature and relative humidity were recorded during the study and the records retained.

[0244] Diet. Animals were fed with LabDiet 5053 rodent diet and water was provided ad libitum.

[0245] Animal Randomization and Allocations: Animals were allocated into six (6) groups at the start of the study: one (1) group of 5, and five (5) groups of 15 mice each. Following assessment of T cell engraftment, animals in which disease was induced were randomized by the percent frequency of peripheral CD4+ T cells. Each animal was identified by an ear punch corresponding to an individual number. A cage card was used to identify each cage and was marked with the study number, treatment group number, and animal numbers.

[0246] Experimental Design: A minimum of 10-14 days prior to the start of the experiment a cohort of animals underwent surgical implantation of a cecal cannula. A sufficient number of animals underwent implantation to allow for 80 cannulated animals to be enrolled in the study.

[0247] Colitis was induced on Day 0 in 75 male RAG2- / - mice by IP injection of 0.5x106CD44- / CD62L+ T cells isolated and purified from C57Bl / 6 recipients. Donor cells were processed as follows. Spleens were harvested from 57 C57Bl / 6 mice and processed / sorted using Miltenyi MACS columns. An additional five mice (Group 1) served as no disease controls. All recipient mice were weighed daily and assessed visually for the presence of diarrhea and / or bloody stool. Disease activity index (DAI) scoring was performed daily from Days 0-42. Cages were changed every two weeks starting on Day 7, with care taken to capture ¼ of dirty cage material for transfer to the new cage. On Day 13, blood was collected via retro-orbital (RO) eye bleed, centrifuged, and plasma stored at -80°C. The pelleted cells were re-suspended in buffer to determine the presence of T cells by FACS analysis of CD45+ / CD4+ events.

[0248] Based on the CD45+ / CD4+ events on Day 13, animals were re-distributed in order to obtain similar CD45+ / CD4+ percentages in each group. Test article treatment with vehicle, NRG-4 parent, or AVEX-1022 began on Day 14 and continued until Day 42 as outlined in Table 1. All treatments were delivered via intracecal (IC) injection daily from Days 14-42 at a volume of 0.075mL / 20g. The animals in Groups 1 and 2 were treated with vehicle. The animals in Group 3 were treated with NRG-4 parent (1500 μg / kg / day), and the animals in Groups 4-6 were treated with three different doses of SEQ ID NO:22 (200, 600 and 1500 μg / kg / day.)4878-9453-5582.4Page 61 of 80079065-000004WOPT

[0249] Mice underwent HD video endoscopy on Days 21, 34, and 42 (before euthanasia) to assess colitis severity. Images were captured from each animal at the most severe region of disease identified during endoscopy. Additionally, stool consistency was scored during endoscopy using the parameters in Table 13-2.

[0250] The animals from all groups were euthanized via CO2inhalation following endoscopy on Day 42 and underwent terminal sample collection. Terminal blood was collected and prepared for plasma. The colon was excised, rinsed, measured, weighed, and then trimmed to 5 cm in length. The most distal and proximal 1.5 cm sections were placed in formalin for subsequent histological evaluation. The middle 2 cm portion were weighed and snap frozen in liquid nitrogen. All plasma and frozen colon tissue were stored at -80°C until shipment to the sponsor or used for selected endpoint analysis.

[0251] Table 13-1. Study Design of Naïve THCell Transfer Mouse Colitis ModelBlood Collection for FACS: Day 13 Engraftment check; Terminal Collections (Day 42): Blood – plasma; Colon – snap frozen; formalin.

[0252] Animal Health Observations and Disease Activity Index (DAI) Score: Animals were observed daily for signs of adverse animal health (e.g., visual inspection of movement, lethargy, normal mouse behaviors), as well as survival, in order to assess possible differences among treatment groups and / or possible toxicity resulting from the treatments. Animals were be observed daily for weight loss, diarrhea, and blood in stool. Each of these parameters was individually scored according to the disease activity index (DAI) scoring scheme shown in Table 13-2, yielding DAI scores with a minimum of 0 (>4.99% body weight loss, no diarrhea, no blood in stool) and a maximum of 13 (>30% body weight loss, diarrhea, and gross bleeding). Any animal that loses >30% of its body weight, shows an inability to eat, or is moribund, was euthanized and did not have samples collected. Table 13-2. Disease Activity Index Scoring Scheme (DAI)4878-9453-5582.4Page 62 of 80079065-000004WOPT

[0253] Body Weight and Survival: Animals were observed daily (weight, morbidity, survival, presence and severity of diarrhea and / or bloody stool) in order to assess possible differences among treatment groups and / or possible toxicity resulting from the treatments. Animal deaths were evaluated during the course of the study. In this model, animal deaths are commonly attributable to severe colitis or perforation during endoscopy. In this study, 15 animals were either found dead or were euthanized due to either euthanasia criteria or moribundity.

[0254] Endoscopy: Each mouse underwent video endoscopy on Days 21, 34, and 42 (prior to euthanasia) using a small animal endoscope (Karl Storz Endoskope, Germany), under isoflurane anesthesia. During each endoscopic procedure, still images as well as video were recorded to evaluate the extent of colitis and the response to treatment. Additionally, an attempt was made to capture an image from each animal at the most severe region of disease identified during endoscopy. Colitis severity was scored using the parameters defined in Table 13-3. Additionally, stool consistency was scored during endoscopy using parameters defined in Table 13-4. Table 13-3. Colitis severity scoring.Table 13-4. Stool consistency scoring.

[0255] Statistical Analyses. Data were evaluated using either one-way ANOVA with Dunnett’s or Holm-Šídák’s multiple comparisons test to compare all groups to the vehicle control group4878-9453-5582.4Page 63 of 80079065-000004WOPTor Kruskal-Wallis test with Dunn’s multiple comparisons test used to compare all groups to the vehicle control group. All statistical analyses were performed using GraphPad Prism 8.3 (La Jolla, CA). Statistical significance was achieved when p<0.05. Results

[0256] Survival: THCell Transfer Mouse Colitis model is associated with a high degree of mortality. Animals were assessed daily for survival or moribundity, and a Kaplan-Meier curve showing survival over the duration of the study is shown in Figure 17 panel A. Disease induction was associated with variable degrees of mortality, though mortality was overall increased in diseased groups relative to naïve animals. Among diseased groups, the highest survival was observed in the 200 μg / kg dose SEQ ID NO:22 treated animals (92.3%, Group 4), whereas the lowest survival was observed in the vehicle treated animals (66.7%, Group 2). Survival in the study groups treated with NRG-4 parent, 600 μg / kg SEQ ID NO:22 dose, and 1500 μg / kg SEQ ID NO:22 dose was equivalent (80%). Figure 17 panel B shows the survival data for parent NRG-4 versus all SEQ ID NO:22 dose groups. The grouped SEQ ID NO:22 treatment cohorts show a clear higher survival trend than the vehicle group and is also seen with the 1500 μg / kg NRG-4 treatment group.

[0257] Weight Loss: Animals in the THCell Transfer Mouse Colitis model lose weight with disease progression. Animals were weighed daily for the duration of the study. The percent body weight change relative to Day 0 was calculated and is shown in Figure 18. To account for changes in average weight change due to attrition contributing to survivor bias, the body weight with which an animal died was carried forward. In order to determine the statistical significance of overall differences in mean percent body weight change between groups, the area under the curve (AUC) was calculated using the trapezoidal rule transformation from Day 0 and is shown in the figure inset. The 600 ^g / kg SEQ ID NO:22 dose showed the best therapeutic response compared to the 1500 ^g / kg NRG-4 and the vehicle treated groups; though this effect did not reach statistical significance (NS, p=0.051).

[0258] The Mean percent weight change comparisons by day were determined using 2-way ANOVA analysis. All treatment groups were compared to the vehicle control group. Animals that were treated with 600 ^g / mL SEQ ID NO:22 demonstrated improvement in weight loss for multiple days over the course of the study (Days 27-32 and Days 35-38).

[0259] Of note, the 600 μg / mg SEQ ID NO:22 dose group was also found to show the best effect in prevention of weight loss in DSS colitis mouse model.

[0260] Composite DAI Scores: Animals were observed daily for weight loss, diarrhea, and blood in stool. Each of these parameters was individually scored on a daily basis according to the disease activity index (DAI) scoring scheme shown in Table 13-2, yielding DAI scores with a minimum of 0 ( İ4.99% body weight loss, no diarrhea, no blood in stool) and a maximum of 12 (>20% body weight loss, diarrhea, and gross bleeding). Composite DAI scores over time are shown in Figure 19 with death scores carried forward. In order to determine the statistical significance of overall differences in mean4878-9453-5582.4Page 64 of 80079065-000004WOPTcomposite DAI score between groups, the area under the curve (AUC) was calculated using the trapezoidal rule transformation from Day 0 and is shown in the figure as well. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test used to compare all groups to the vehicle control Group 2. Data is presented as mean ± SEM. n=13-15 per group.

[0261] Figure 19 shows the DAI score comparison of the 600 μg / kg SEQ ID NO:22 and NRG- 4 dose groups. Diseased animals demonstrated increased DAI scores when compared to naïve animals by AUC analysis. A 48% decrease in cumulative DAI scoring was observed with SEQ ID NO:22 treatment (600 ^g / kg) compared to the vehicle control group, while treatment with NRG- 4 parent resulted in only a 14% decrease compared to the control.

[0262] Endoscopy and Stool Consistency. Colonic inflammation was evaluated via endoscopy on Days 21, 34, and 42. Colitis was scored visually on a five-point scale that ranges from 0 for normal to 4 for severe ulceration; stool consistency was likewise scored visually on a five-point scale that ranges from 0 for normal to 4 for bloody diarrhea (see Tables 13-3 and 13-4). Animals in which colitis was induced by T cell transfer demonstrated increased mean colitis severity and stool consistency scores at all timepoints; mean colitis severity scores were significantly lower in naïve animals as compared to diseased animals treated with vehicle on Days 34 (p<0.05) and Day 42 (p<0.05). No major differences in Day 34 or Day 42 colitis severity scores were observed when comparing between treatment groups. Day 21 colitis severity scores were decreased in diseased animals treated with 200 ^g / kg SEQ ID NO:22 (26% decrease) relative to vehicle and a 7% reduction in the mean colitis severity score compared to the vehicle control group was observed in the animals treated with both NRG-4 parent and 600 ^g / kg SEQ ID NO:22 (Figure 20). However, these differences were not statistically significant.

[0263] Mean stool consistency scores were similar among treatment groups at the Day 21 endoscopy. On Day 34, treatment with all test articles except for 1500 ug / kg SEQ ID NO:22 resulted in a slight increase in the mean stool consistency scores compared to the vehicle control group. On Day 42, treatment with the test articles resulted in an overall decrease in the mean stool consistency scores compared to the vehicle control group, where 1500 ug / kg SEQ ID NO:22 treatment caused the largest decrease (41.5%) (Figure 21).

[0264] Colon Weight:Length Ratio: All surviving animals were euthanized on Day 42 for terminal collections. The colon was excised, measured, rinsed, weighed, and the colon weight:length ratio was calculated. Due to likely survival bias at the study termination on Day 42, there was no difference observed between the mean colon weight:length ratios of the naïve animals compared to the vehicle control animals. There were no significant differences between test-article-treated animals and vehicle-treated animals.

[0265] Colon Histopathology Fixed colon samples were processed for embedding, staining with H&E, and pathology evaluation. The proximal and distal segments of the collected colon were scored individually. Lesions were given a severity score of 0-5, where 0=not present / normal,4878-9453-5582.4Page 65 of 80079065-000004WOPT1=minimal, 2=mild, 3=moderate, 4=marked, and 5=severe). Inflammation, necrosis / gland loss, erosion, hyperplasia, and edema scores were evaluated and added together to determine a sum colitis score for each sample (range 0-25).

[0266] Histopathologic findings were consistent with those expected from the naïve TH cell- induced model of colitis, including the development of inflammation, gland loss, erosions, submucosal edema, and epithelial hyperplasia. Survival bias was present in the histological data as the animals with the most severe disease, particularly in the vehicle control group, succumbed prior to planned terminal sample collection at Day 42. In this study, while administration of SEQ ID NO:22 (referred as TA in Figure 22A and 22B) at any administered dose was not associated with reductions in overall colitis severity that achieved statistical significance in either the proximal or distal colon, there were slight reductions in colitis severity that trended with increased dose of SEQ ID NO:22. Erosion and edema in the proximal colon, as determined by semi-quantitative histological scoring, were reduced following treatment with SEQ ID NO:22 compared to the vehicle control, though this difference was not statistically significant (Figure 22A). In the distal colon, more prominent reductions in edema and erosions were noted at the two higher administered doses (600 μg / kg, Group 5 and 1500 μg / kg, Group 6) of SEQ ID NO:22 (“TA”) (Fig.22B).

[0267] We have demonstrated that intra-cecally administered SEQ ID NO:22 is efficacious in the Naïve THCell Transfer Mouse Colitis Model by the following criteria: (1) improved survival (2) decreased body weight loss, (3) decreased DAI scores and (4) despite the increased mortality associated the model and hence the confound of survival bias, SEQ ID NO:22 treatments results in decreased levels of edema and erosions in the proximal and distal colon. Combined with the efficacious result in the intra-cecally delivered SEQ ID NO:22 in the DSS mouse colitis model, we have demonstrated compelling evidence of GI protective effects of the NRG4 analogs in two very different mouse colitis models. Example 6 Development of Enteric-Coated NRG-4-Microcystalline Cellulose Beads

[0268] This procedure describes an exemplary process to make enteric-coated NRG-4- microcystalline cellulose (MCC) beads for the targeted delivery of the lead NRG-4 analogs (e.g., those identified in Example 1) in the intestinal tract, denoted as “NRG-4-MCC beads”. These coated MCC beads are loaded into gelatin capsules for the eventual drug product.

[0269] Peptidic analogs of NRG-4 delivered in enterically coated MCC beads (filled in gelatin capsules) are gut-localized, upon oral intake, and have no systemic exposure.

[0270] In one embodiment, the NRG-4-MCC beads are protected as they pass through the stomach by an acid-stable EUDRAGIT® L 30 D-55 polymer coating. The dissolution of the enteric coat under near-neutral pH conditions in the intestine, followed by MCC bead disintegration, results in4878-9453-5582.4Page 66 of 80079065-000004WOPTa slow and sustained release of the polypeptide. In some embodiments, enteric coated NRG-4-MCC bead suspensions are orally gavaged in mice. In some embodiments, gelatin capsules each containing a quantity of enterically coated NRG-4-MCC beads are orally ingested or administered to the mouth in mice.

[0271] Figure 16A shows an exemplary manufacturing workflow. In some embodiments, each batch size is about 100 g (total weight) to minimize consumption of NRG-4 analog, and a targeted drug load of 0.1-0.5 wt % load. This entails about 100 mg-500 mg of active pharmaceutical ingredient (API) for each batch. MCC spheres are used as the substrate to which the NRG-4 analog are applied in the granulating solution. A wet binding agent is also incorporated into the solution. Prior to manufacturing, solution stability is evaluated and established on a very small scale to ensure the analog is compatible with the binder. To minimize degradation of the NRG-4 analog, minimal inlet temperatures is employed during processing, and spray rates is slowed over a longer period of time for both active layering and enteric layer application.

[0272] In order to confirm the stability of the NRG-4 analog throughout the process, sampling is conducted at multiple time points at the following intervals: granulating solution, during coating of MCC substrate, and enteric coating of active spheres with Eudragit. The various enteric coat levels are evaluated for acid resistance in appropriate media (pH 4 buffer), and to ensure coat levels are sufficient to achieve the targeted release profile (>pH 5.5). Three batches are prepared at this scale in an evolutionary manner. The results from the first batch are used to guide appropriate process and formulation changes for the second batch and same strategy for the 3rditeration. In addition, enteric- coated MCCs without any NRG-4 are prepared to serve as vehicle control for the efficacy study below and for safety assessment study in vivo. Preparation and Characterization of Enteric Coated NRG-4-Microcystalline Cellulose Beads.

[0273] Capsules filled with enteric-coated peptidic NRG-4 analog-microcrystalline cellulose (MCC) beads were manufactured for targeted delivery of NRG-4 peptidic analogs (e.g., SEQ ID NO:22) in the intestines. EUDRAGIT® L 30 D-55 polymer coating technology was used for an acid-stable delivery of the SEQ ID NO:22-MCC beads, allowing SEQ ID NO:22-MCC beads passage through the stomach and dissolution in the near-neutral intestinal pH. After the enteric coating has disintegrated, SEQ ID NO:22 is released for targeted drug delivery to the intestines.

[0274] The process for manufacturing enteric-coated Peptidic NRG-4 analog-MCC beads includes coating of MCC beads with a polypeptide (a peptidic NRG-4 analog disclosed herein, e.g., one having an amino acid sequence of SEQ ID NO:22) drug substance then coating the Peptidic NRG-4 analog-MCC beads with EUDRAGIT® L 30 D-55 via fluid bed granulator. For this process, an excipient compatibility study was completed to determine the excipient that would be used to manufacture the finished product. The compatibility assay determined if there was any impact to the stability of peptidic NRG-4 analog drug substance by different excipients. MCC and Hydroxypropyl4878-9453-5582.4Page 67 of 80079065-000004WOPTCellulose were found to exhibit minimal impact on active pharmaceutical ingredient (API) stability through the 8-week excipient compatibility study and were selected to be used for manufacture.

[0275] A Freund Vector VFC-Lab Micro fluid bed granulator with a Würster column insert was used to coat the MCC beads with the SEQ ID NO:22 as well as the EUDRAGIT® L 30 D-55. First, the MCC beads were coated with an aqueous solution consisting of 1.5% w / v hydroxypropyl cellulose and 0.2% w / v SEQ ID NO:22 in water. Then the SEQ ID NO:22-MCC beads were enterically coated with 5% w / v EUDRAGIT® L 30 D-55 aqueous suspension, creating the enteric-coated SEQ ID NO:22- MCC finished product.

[0276] The optimal processing parameters for manufacturing were determined through a generational approach. The first generation determined the efficiency of applying the SEQ ID NO:22 drug substance to the MCC beads to a target of 0.1 % w / w, shown to be successful through analytical results. In generation II, API drug load was increased from 0.1% w / w to 0.12% w / w and the process parameters were optimized. Generation III manufacture yielded approximately 200 g of enteric-coated SEQ ID NO:22 MCC beads with a final drug load of 0.166% w / w as determined by HPLC analysis. Placebo beads, i.e. enteric-coated MCC beads with no SEQ ID NO:22 were manufactured with the same process. Table 14. Concentration of SEQ ID NO:22 in drug-loaded enteric-coated MCC Beads.

[0277] The release of SEQ ID NO:22 from these enteric-coated MCC beads was determined by incubation and paddle stirring at 200 rpm of 2.5 gm of beads in 75 ml of buffer under different pH conditions, followed by filtration through a 0.2 micron nylon filter and HPLC analysis. The dissolved values reported in Table 15 were based on the reported drug load concentration of 1660 ug / g. The data indicate no release of SEQ ID NO:22 at pH 4.00, whereas >95% of SEQ ID NO:22 is released at pH 6.00 by 1 hour.

[0278] Stability testing on generation III finished product was conducted at 0-, 1-, 2-, and 3- week time points. Results showed there was minimal product degradation at the 25°C / 60% RH, but with increased degradation at the 40°C / 75% RH (Figure 23). To minimize product degradation in storage, the finished product will be stored at 2-8°C.

[0279] Oral gavage administration may not be successful with the bead suspension due to inconsistent dosing arising from clogging of the gavage needle applicator. Given the relatively high density of the beads, a viscous solution was required to keep the beads in suspension and to facilitate consistent administration. Since hydroxypropyl cellulose is already used in the formulation, it was selected as the thickening agent for the suspension. To determine the concentration of the thickening4878-9453-5582.4Page 68 of 80079065-000004WOPTagent (e.g., hydroxypropyl cellulose) required for suitable administration, concentrations of 0%, 5%, 10%, and 20% w / v hydroxypropyl cellulose in citrate-phosphate buffer pH 4 were evaluated. Based on flocculation rate and viscosity of the enteric-coated SEQ ID NO:22-MCC beads, 15% w / v hydroxypropyl cellulose solution was selected. However, viscosity and thickness of the bead suspension may clog gavage needle applicator.

[0280] As an alternative oral delivery method, enteric coated SEQ ID NO:22-MCC beads were encapsulated into size M gelatin capsules. Preliminary studies showed a few instances of premature separation of the shell from the body of the capsule prior to administration. To mitigate capsule separation, capsules were sealed with a 50% ethanol solution. Feasibility studies on the oral administration of these sealed capsules in mice were successful.

[0281] Gelatin capsules filled with enteric coated, NRG-4 peptidic analog-MCC beads, will be studied for efficacy in the mouse DSS colitis model using oral dosing of drug loaded and vehicle loaded M capsules and using anti-p40 mAb as a positive efficacy control. The study will be conducted using male and female mice to address Sex As a Biological Variable; equal numbers of age and sex- matched mice will be randomly allocated to study groups. Table 15. SEQ ID NO:22 release from enteric coated MCC beads at different pH conditions. ND = Not detectable. RSD = relative standard deviation. % Dissolved SEQ ID NO:22Table 16. An exemplary PK / PD Study Design with NRG-4-MCC beads in DSS Colitis Mice.4878-9453-5582.4Page 69 of 80079065-000004WOPT

[0282] Next, an animal study with enteric-coated NRG-4 analog-MCC beads is conducted to confirm that an NRG-4 analog is indeed delivered to the colon and to demonstrate efficacy in the mouse dextran sulfate sodium (DSS)-induced colitis model. To increase reproducibility and to address Sex As a Biological Variable, equal numbers of age and sex-matched mice, randomly allocated to study groups, is used (Table 13). Group 1, consisting of 6 male and 6 female C57Bl / 6 mice, serve as a no-disease control. Colitis is induced in 60 male and 60 female mice (Groups 2-7) by exposure to 3% DSS-treated drinking water from Days 0 to 5. NRG-4-MCC beads are delivered daily via gavage as a suspension in acidic pH formulation from Days 6-19. A 5 mL / kg dose volume of the NRG-4-MCC bead suspension, with the appropriate bead amount based on NRG-4 load to account for three predetermined drug doses. Mouse anti-IL-12 p40 (BioXCell), a reliable positive control in Biomodels’ studies, is used as a comparator and is administered IP once a day on Days 6, 9, 12, 15 and 18. Tissue collection procedures and efficacy measures are then carried out. Briefly, each mouse undergoes video endoscopy to assess the severity of colitis using a small animal endoscope (Karl Storz Endoskope, Germany) while under isoflurane anesthesia. During each endoscopic procedure, still images as well as video are recorded to evaluate the extent of colitis and the response to treatment. Additionally, an image from each animal at the most severe region of disease identified during endoscopy is captured. This additional measure of colitis severity is scored (examiner blinded to treatment groups) using a 0-4 scale (0= normal; 1= loss of vascularity; 2= loss of vascularity and friability; 3= friability and erosions; 4= ulcerations and bleeding). As an additional secondary endpoint, stool consistency is scored during the endoscopy also using a 0-4 scale (0= normal, well-formed pellet; 1= loose stool, soft, staying in shape; 2= loose stool, abnormal form with excess moisture; 3= watery or diarrhea; 4= bloody diarrhea). Feces is collected and frozen to allow for possible subsequent assessment of lipocalin-2 as an inflammation biomarker. Following endoscopy, animals from all groups are euthanized by CO2 inhalation and will undergo terminal blood and colon collection.4878-9453-5582.4Page 70 of 80079065-000004WOPT

[0283] Statistical analysis of the data is carried out by one-way ANOVA with Dunnett’s multiple comparisons test used to compare NRG-4 and anti-IL-12 p40 dose groups to their respective vehicle controls. All statistical analyses are performed using GraphPad Prism 8. Group sizes chosen for the study provide 90% power to detect at least a 25% reduction of the variables compared to vehicle control (ANOVA, set at 0.05).

[0284] In addition, levels of the delivered polypeptide(s) are assayed in terminal plasma samples and in colon homogenate from the dosed groups. Mass spectrometry-based bioanalytical assays are conducted for detection of the polypeptide(s) in both plasma and the colon. This assay has a lower limit of quantitation (LLOQ) of ~ 1-2 ng / mL in plasma. For the colon specimens, 1 cm colon sections from the animal studies are homogenized using an Omni Bead Ruptor. The polypeptides (NRG-4 analogs) are immunocaptured from plasma or colonic homogenate extracts from either polypeptide (NRG-4 analogs) treated animals or from plasma / colonic extracts spiked with polypeptides (NRG-4 analogs), disulfide bonds will be reduced with DTT, and then the peptide are digested using trypsin. Digested samples are extracted using reversed phase solid phase extraction (SPE) cartridges and analyzed by RPLC-MS / MS using a Waters BEH 300 C18 column for separation from interferants. Peptide fragments that are unique among the human proteome sequences currently available on neXtProt76 are selected for reaction monitoring and peptide transitions. Absolute quantification is achieved using isotopically labeled peptides corresponding to the transition peptides. It should be noted that metabolic stability studies with parent NRG-4 revealed that trypsin only cleaves at Lys. Thus, this permits synthesis of a stable isotope labeled reference peptide fragment using lysine (13C6 or13C6,15N2) or arginine (13C6 or13C6,15N4) to substitute for either of these residues preserved in the disulfide domain of the optimized NRG-4 analog. Additionally, tissue sections of fixed colon tissues from the NRG-4 dosed and vehicle groups are analyzed by immunohistochemistry for Akt phosphorylation to confirm ErbB4 receptor activation. Sections are analyzed by whole slide digital imaging and quantified using Reveal’s proprietary software, IMAGEDXTM, to measure the number of positive cells and staining intensity.

[0285] LC / MS / MS techniques were developed for the determination of polypeptides (NRG-4 analogs) in mammalian plasma and colon. For example, a protein precipitation (PPT) extraction procedure was developed to extract the polypeptide (NRG-4 analog) from the plasma and tissue matrix. An aliquot of the extract was injected onto a LC / MS / MS triple quadrupole mass spectrometer. A Pursuit Diphenyl, 5 LC column (2.1 x 100 mm) was used to separate the polypeptide (NRG-4 analog) and the internal standard, stable-labeled polypeptide (NRG-4 analog), from interfering compounds that may be present in the sample. The peak area of the product ion of the compound was measured against the peak area of the product ion of the internal standard. A calibration curve ranging from 5.00 to 2000 ng / mL (ng / g for tissues) was used to quantify the polypeptide (NRG-4 analog) in the samples.4878-9453-5582.4Page 71 of 80079065-000004WOPT

[0286] Accuracy and precision were evaluated by creating calibration curves with the standards and using the derived equation from the weighted linear regression line of peak area ratios to generate the calculated values. The accuracy for the back-calculated standard curve points is within ± 15% of the nominal value except at the LLOQ, where it should not deviate by more than ± 20% to be considered acceptable. At least ¾ of the calibration standards in each batch should meet this accuracy criterion, including the LLOQ and ULOQ, in order for the batch to be considered acceptable. Both the plasma (r = 0.9974) and colon (r = 0.9958) calibration curves met this criterion.

[0287] The mean value of the accuracy the QC samples should be within ± 15% of the nominal value to be considered acceptable. Both the plasma and colon QCs met this criterion.

[0288] Selectivity: Blanks analyzed during method development indicated no significant interfering peaks are present at the retention time of SEQ ID NO:22 in the plasma or colon extracts.

[0289] Hence, the developed technique was accurate and precise and can be used to determine the polypeptides (NRG-4 analog, e.g., SEQ ID NO:22) in mammalian plasma and colon samples.

[0290] We also achieved multi-gram scale production by Solid Support Peptide Synthesis (SPPS). Figure 24 shows the characterization of SEQ ID NO:22, which is a 46-amino acid long polypeptide, having 3 disulfide bonds. We also have scalable SPPS process in place to obtain kilogram quantities; and fragment condensation approach may further improve production process and reduce cost. We also have systems for recombinant polypeptide production.

[0291] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0292] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.4878-9453-5582.4Page 72 of 80079065-000004WOPT

[0293] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

[0294] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.”4878-9453-5582.4Page 73 of 80079065-000004WOPT

Claims

WHAT IS CLAIMED IS:

1. An enteric coated composition comprising a core in the form of a particle and an enteric coating for the core, wherein the particle comprises a polypeptide and microcrystalline cellulose, and wherein the enteric coating imparts protection to the core so that the core is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher, said polypeptide comprising an amino acid having Formula I: (Formula I): Xaa0- [Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6Xaa7Xaa8Xaa9Xaa10Xaa11Xaa12Xaa13Xaa14Xaa15Xaa16Xaa17Xaa18Xaa19Xaa20Xaa21Xaa22Xaa23Xaa24Xaa25Xaa26Xaa27Xaa28Xaa29Xaa30Xaa31Xaa32Xaa33Xaa34Xaor a variant of Formula I having at least 90% sequence identity to Formula I, wherein: Xaa0is absent or selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO:47), and (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is S, Q, A, or E and n=1-5, and Xaa0is optionally further modified with a first hydrophobic moiety; Xaa1is absent; Xaa2is absent or Lys (K); Xaa3is absent or Gly (G) or Thr (T) or Ser (S); Xaa4is absent or Asp (D) or Glu (E) or Asn (N) or Gln (Q); Xaa5is His (H); Xaa6is Glu (E) or Asp (D); Xaa7is Glu (E) or Gln (Q); Xaa8is Pro (P); Xaa9and Xaa23are Cys (C), which are capable of forming a disulfide bond; Xaa10is Gly (G); Xaa11is Pro (P); Xaa12is Ser (S); Xaa13is His (H); Xaa14is Lys (K) or Arg (R); Xaa15is Ser (S); Xaa16is Phe (F) or Tyr (Y); Xaa17and Xaa34are Cys (C), which are capable of forming a disulfide bond; Xaa18is Leu (L), Ile (I), or Val (V); 4878-9453-5582.4 Page 74 of 80079065-000004WOPTXaa19is Asn (N), Glu (Q), Lys (K), Arg (R), or His (H), wherein if Xaa19is His, then Xaa47is not Gln (Q); Xaa20is Gly (G), Ala (A), Ser (S), Asn (N), Gln (Q), Glu (E) or Asp (D), wherein if Xaa19Xaa20is His-Asp, then Xaa47is not Gln (Q); Xaa21is Gly (G); Xaa22is Leu (L); Xaa24is Tyr (Y); Xaa25is Val (V); Xaa26is Ile (I); Xaa27is Pro (P); Xaa28is Thr (T); Xaa29is Ile (I) or Leu (L); Xaa30is Pro (P); Xaa31is Ser (S) or Gly (G); Xaa32is Pro (P); Xaa33is Phe (F); Xaa35is Arg (R); Xaa36and Xaa45are Cys (C), which are capable of forming a disulfide bond; Xaa37is Val (V) or Ile (I); Xaa38is Glu (E); Xaa39is Asn (N); Xaa40is Tyr (Y); Xaa41is Thr (T); Xaa42is Gly (G); Xaa43is Ala (A); Xaa44is Arg (R); Xaa46Xaa47is Glu-Glu, Asp-Gln, Asp-Asn, or Glu-Gln, and Xaa48Xaa49is Val-Phe or Phe-Leu, wherein if Xaa46Xaa47is Glu-Glu, then Xaa48Xaa49is Val-Phe, if Xaa46Xaa47is Asp-Gln, then Xaa48Xaa49is Val-Phe or Phe-Leu, if Xaa46Xaa47is Asp-Asn, then Xaa48Xaa49is Val-Phe, and if Xaa46Xaa47is Glu-Gln, then Xaa48Xaa49is Val-Phe; Xaa50is Leu (L); and Xaaμis absent, or selected from the group consisting of: PGS, S,4878-9453-5582.4Page 75 of 80079065-000004WOPTGS, GGS, GGGS (SEQ ID NO:47), (GGGGXaa^)n(SEQ ID NO:43) wherein Xaa^is S, Q, A, or E and n=1-5, Pro^Gly^Ser^Lys J-Glu ([SEQ ID NO:44] J-Glu), Ser^Lys J-Glu, Gly^Ser^Lys J-Glu, Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:45] J-Glu), Gly^Gly^Gly^Ser^Lys J-Glu ([SEQ ID NO:46] J-Glu), and a combination of (GGGGXaa^)n(SEQ ID NO:43) and Lys J-Glu, wherein the bonds depicted by represent an amide linkage between İ-amino group of the Lys and Ȗ-carboxyl group of the Glu, optionally said Glu being further bound to a second hydrophobic moiety; wherein if Xaa0is absent, the N-terminus the polypeptide is optionally modified with a hydrophobic moiety, and wherein if Xaaμis absent, the C-terminus the polypeptide is optionally modified with a hydrophobic moiety; and wherein the variant of Formula I has at least one biochemical activity exhibited by a polypeptide selected from the group consisting of Formula I and SEQ ID NO:1, and optionally wherein the polypeptide is not any one of human neuregulin (NRG) 4 (NRG-4) (5- 50), human NRG-4(1-62), epidermal growth factor (EGF), human NRG-3, rat NRG-4, mouse NRG-4, gorilla NRG-4, and chimpanzee NRG-4.

2. An enteric coated composition in the form of a tablet, comprising an enteric coating on a compressed formulation, wherein the compressed formulation comprises a polypeptide, microcrystalline cellulose, and optionally a binding agent, and wherein the enteric coating imparts protection to the core so that the core is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher, said polypeptide comprising an amino acid having a same Formula I as that of the polypeptide in the composition of claim 1, and said enteric coating comprising a copolymer derived from methacrylic acid and ethyl acrylate.

3. The composition of claim 1, wherein the particle further comprises a binding agent selected from the group consisting of hydroxypropyl cellulose, calcium chloride, polyvinyl alcohol, hypromellose, magnesium stearate, and a combination thereof.

4. The composition of claim 1, wherein the enteric coating comprises a copolymer derived from methacrylic acid and ethyl acrylate.4878-9453-5582.4Page 76 of 80079065-000004WOPT5. The composition of claim 1, wherein the polypeptide is at least 0.05% by weight of the enteric coated composition.

6. The composition of claim 1, further comprising one or more excipients.

7. The composition of any one of claims 1 and 3-6, wherein the core is in the form of a microparticle or a nanoparticle.

8. A dosage form of a pharmaceutical composition, comprising a shell and a plurality of the enteric coated composition of any one of claims 1 and 3-7.

9. The dosage form of claim 8, in the form of a capsule and wherein the shell comprises gelatin.

10. The composition of claim 1 or 2, wherein the biochemical activity exhibited by the polypeptide selected from the group consisting of Formula I and SEQ ID NO:1 comprises: inducing homodimerization of ErbB4 subunits, having specificity in inducing homodimerization of ErbB4 subunits over homodimerization of EGFR subunits by a difference in EC50of at least 1,000 fold, having at least 90% stability, or more stable, compared to neuregulin-4, with respect to resisting enzymatic degradation in intestinal fluid or a simulated intestinal fluid, having at least 90% stability, or more stable, compared to neuregulin-4, with respect to resisting enzymatic degradation in gastric fluid or a simulated gastric fluid, or a combination thereof.

11. The composition of claim 10, wherein the stability of the polypeptide is with respect to resisting degradation by chymotrypsin or trypsin in the intestinal fluid or the simulated intestinal fluid, and resisting degradation by pepsin in the gastric fluid or the simulated gastric fluid, and wherein the simulated intestinal fluid comprises a phosphate buffer at a pH of about 6.8, and the simulated gastric fluid comprises a solution of sodium chloride at a pH of about 1.

2.

12. The composition of claims 1 or 2, wherein the first hydrophobic moiety and the second hydrophobic moiety, if present, independently comprises a lipid moiety; a moiety having a structure of HO2CíRíC(=O)í, where R is C3-C28 alkane, alkene, or alkyne; or a polymer other than an amino acid polymer.

13. The composition of claim 1 or 2, wherein both X0and Xμare absent.

14. The composition of claim 1 or 2, wherein only one of the first hydrophobic moiety and the second hydrophobic moiety is present in the polypeptide.

15. The composition of claim 14, wherein the one present hydrophobic moiety is selected from palmitoyl, tetradecanoyl, or a C6-C12 or C18-C26 fatty acyl group.

16. The composition of claim 12, wherein the one present hydrophobic moiety is derived from hexadecanedioic acid or tetradecanedioic acid.

17. The composition of claim 1 or 2, wherein if Xaa0, Xaa1, and Xaa2are absent, if Xaa3is G, and the N-terminus is modified with a hydrophobic moiety, then G is modified with the4878-9453-5582.4Page 77 of 80079065-000004WOPThydrophobic moiety C=O(CH2)nR, where n can be an integer from 12-22 and R is -CO2H, - CO2CH3, -CO2NH2, -CO2NHCH3, -CO2N(CH3)2, -CH3, or -NH2.

18. The composition of claim 1 or 2, wherein the polypeptide is selected from the group consisting of SEQ ID NOs:7-10, 15-17, 21, and 22.

19. The composition of claim 1 or 2, wherein the polypeptide is selected from SEQ ID NOs:8, 15- 17, 21, and 22.

20. The composition of claim 1 or 2, wherein the polypeptide is selected from the group consisting of SEQ ID NOs:48-53.

21. The composition of claim 1 or 2, wherein the polypeptide is a variant of Formula I having at least a fragment comprising three amino acids corresponding to the Xaa48through the Xaa50of SEQ ID NO:42 in sequence alignment with SEQ ID NO:

42.

22. The composition of claim 21, wherein a residue corresponding to the Xaa50of SEQ ID NO:42 is not Pro (P).

23. The composition of claim 22, wherein a residue corresponding to the Xaa24of SEQ ID NO:42 is not Gln (Q) or Val (V).

24. The composition of claim 21, wherein if residues corresponding to the Xaa46through the Xaa49of SEQ ID NO:42 are Asp-Gln-Val-Phe (SEQ ID NO:37), then a residue corresponding to the Xaa50of SEQ ID NO:42 is Leu (L).

25. The composition of claim 21-24, wherein, in the polypeptide, residues corresponding to the Xaa9and the Xaa23of SEQ ID NO:42 are Cys and capable of forming a disulfide bond; residues corresponding to the Xaa17and the Xaa34of SEQ ID NO:42 are Cys and capable of forming a disulfide bond; and residues corresponding to the Xaa36and the Xaa45of SEQ ID NO:42 are Cys and capable of forming a disulfide bond.

26. The composition of any one of claims 1, 2, 10-17, and 21-25, wherein, in the polypeptide, the Xaa46through the Xaa50of SEQ ID NO:42 are not D-enantiomeric amino acids, or wherein residues of the variant, if present, corresponding in sequence alignment to the Xaa46through the Xaa50of SEQ ID NO:42 are not D-enantiomeric amino acids.

27. The composition of any one of claims 1, 2, 10, 11, and 21-25, wherein, in the polypeptide, if the Xaa46through the Xaa50of SEQ ID NO:42, or residues of the variant corresponding in sequence alignment to the Xaa46through the Xaa50of SEQ ID NO:42, are Asp-Gln-Val-Phe- Leu (SEQ ID NO:41), and if the Xaa19of SEQ ID NO:42, or residue of the variant corresponding to the Xaa19of SEQ ID NO:42, is His, then the polypeptide does not comprise a hydrophobic moiety covalently linked to the N-terminus of the polypeptide.

28. A method of preparing the composition of claim 1, comprising: mixing the polypeptide, the microcrystalline cellulose, and a binding agent in a slurry; generating particles from the slurry by granulation, extrusion, and / or spheronization; and4878-9453-5582.4Page 78 of 80079065-000004WOPTspraying coating a pH-sensitive polymer on the particles, so as to obtain the composition of claim 1, wherein the pH-sensitive polymer imparts protection to the particles so that the particles are afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher.

29. A method of preparing the composition of claim 2, comprising: mixing the polypeptide, the microcrystalline cellulose, and the binding agent in a slurry; compressing the slurry in a form of a tablet, thereby forming a compressed formulation; and spraying a pH-sensitive polymer on the tablet, or dipping the tablet in a solution or suspension of the pH-sensitive polymer, so as to form a coating comprising the pH-sensitive polymer for the tablet and thereby obtaining the composition of claim 2, wherein the pH-sensitive polymer imparts protection to the tablet so that the tablet is afforded protection in a low pH environment of 3 or less while capable of releasing the polypeptide at a pH of 5.5 or higher.

30. A method for treating a subject with an inflammatory condition in gastrointestinal tract, comprising administering a pharmaceutical composition of claim 8 to the subject.

31. The method of claim 30, comprising orally administering the pharmaceutical composition and releasing the polypeptide in the subject’s colon.

32. The method of claim 30, wherein the inflammatory condition comprises ulcerative colitis, Crohn’s disease, necrotizing enterocolitis, or radiation-induced gastrointestinal injury.

33. A method for treating a subject with acute lung injury or a metabolic disorder, comprising administering a pharmaceutical composition of claim 8 to the subject.

34. The method of claim 33, wherein the metabolic disorder comprises diabetes or non-alcoholic steatohepatitis.

35. The method of claim 33, wherein the pharmaceutical composition further comprises a permeation enhancer, and the administration is a systemic administration.

36. A method for treating a subject with an inflammatory condition in gastrointestinal tract, comprising orally administering the composition of claim 2 in the form of a tablet to the subject, and releasing the polypeptide in the subject’s colon, wherein optionally the inflammatory condition comprises ulcerative colitis, Crohn’s disease, necrotizing enterocolitis, or radiation- induced gastrointestinal injury.

37. A method for treating a subject with acute lung injury or a metabolic disorder, comprising administering the composition of claim 2 in the form of a tablet to the subject, wherein optionally the metabolic disorder comprises diabetes or non-alcoholic steatohepatitis.

38. The method of any one of claims 30-37, wherein the subject is a human, and wherein the administration provides for a dose of 0.25 mg – 50 mg of the polypeptide, per day, in the administered composition.4878-9453-5582.4Page 79 of 80079065-000004WOPT

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